Rhodium metalloinsertors are octahedral complexes developed to selectively target the mismatches and insertions/deletions (indels) that result from mismatch repair (MMR) deficient cancers. By incorporating particularly wide, aromatic, inserting ligands, these complexes are able to detect thermodynamically destabilized mismatch sites via a binding mode known as metalloinsertion, in which the inserting ligand binds DNA via the minor groove and results in ejection of the destabilized mismatched base pair. In vitro analyses of metalloinsertors have found that these complexes are selectively cytotoxic towards MMR-deficient cancer cells compared to MMR-proficient cells. Furthermore, the newest family of Rh-O metalloinsertors, which includes [Rh(phen)(chrysi)(PPO)]\u00b2\u207a (Rh-PPO), displays preferential cytotoxicities in the nanomolar range, which is significantly more potent than first generation metalloinsertors and many standard of care chemotherapeutics. Given the high level of potency and selectivity of Rh-O metalloinsertors, further clinical development of these complexes has been pursued.
\r\n\r\nHere, we present the first preclinical mouse evaluation of a rhodium metalloinsertor as an anticancer agent. The Rh-O metalloinsertor Rh-PPO was evaluated in the HCT116 colorectal cancer xenograft tumor model alongside saline and oxaliplatin controls. Intraperitoneal studies with Rh-PPO showed significant decreases in tumor volumes over time and final tumor weights, indicating Rh-PPO has notable anticancer activity. Additionally, Rh-PPO treatment resulted in a noteworthy increase in the length of mouse survival that was on par with the FDA approved chemotherapeutic oxaliplatin. Pharmacokinetic analyses revealed rapid absorption of Rh-PPO in plasma with notable accumulation in the liver compared to tumors. Importantly, intratumoral metalloinsertor administration resulted in enhanced anticancer effects, which points to a need for more selective delivery methods in order to further metalloinsertor development.
\r\n\r\nIn order to target cancerous cells with still higher selectivity, routes to metalloinsertor antibody drug conjugate (ADC) designs were explored. By attaching Rh-O metalloinsertors to an antibody specific to cancer-associated antigens, our complexes may become even more specifically directed to induce selective cytotoxicity in diseased cells. Three ADC drug linkers that incorporate maleimide groups into the N^O coordinating ligand of a Rh-O metalloinsertor were designed, synthesized, and characterized. These complexes were evaluated for their cellular potency and selectivity toward MMR-deficient cancer cells. Studies revealed that functionalization of the hydroxyl-containing ancillary ligand resulted in decreased potency and abolished preferential cytotoxicity, contrary to previous studies that assessed modifications of this ligand.
\r\n\r\nLiposomal formulations of Rh-PPO were also explored to further target metalloinsertors to malignant cells. Liposomal drug encapsulations have a demonstrated ability to decrease systemic toxicity and increase tumor drug uptake; therefore, the biological activity of Rh-PPO liposomal formulations was explored. Four distinct Rh-PPO liposome formation methods were developed and the resulting liposomes were assessed for their encapsulation efficiency, cellular toxicity, and stability. Remote loaded Rh-PPO liposomes were found to display the most promising chemical and biological characteristics, although additional optimization of encapsulation procedures is necessary for further preclinical evaluation of this metalloinsertor drug delivery approach.
\r\n\r\nAs metalloinsertors continue preclinical assessment and development, a greater understanding of their mechanism of action is imperative. Biological studies with Rh-PPO and the fluorescent analogue RhPPO-Cy3 have shown that DNA damage from metalloinsertor treatment involves the formation of DNA double strand breaks near metalloinsertor-mismatch binding sites. Furthermore, the DNA damage response, including recruitment of pH2AX and Rad51 proteins, becomes activated in response to Rh-PPO treatment. In order to further elucidate the unique mechanism of action of Rh-O metalloinsertors, which involves both metalloinsertor enantiomers binding to DNA mismatches and displaying biological activity, structural studies are ongoing. X-ray crystallography and microelectron diffraction (microED) techniques have been used in attempts to obtain a high resolution structure of Rh-O metalloinsertors bound to DNA mismatch sites. Gaining these structural insights will be critical to understanding the increased cytotoxic selectivity and uniquely high potency of these second generation metalloinsertor complexes.
\r\n\r\nThe experiments detailed in this thesis have advanced the preclinical development of rhodium metalloinsertors. The ability of Rh-O metalloinsertors to decrease tumor growth in vivo has been established. Additionally, liposomal and ADC metalloinsertor drug formulations have been pursued as drug delivery systems, and the biological mechanisms relevant to metalloinsertor activity have been analyzed. Additional efforts to study rhodium metalloinsertors will continue to advance these promising chemotherapeutics as novel, targeted treatments for MMR-deficient cancers.
", "doi": "10.7907/dmqv-ed54", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13800", "collection": "thesis", "collection_id": "13800", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082020-163907557", "primary_object_url": { "basename": "Silva_RebekahMB_2020.pdf", "content": "final", "filesize": 21105463, "license": "other", "mime_type": "application/pdf", "url": "/13800/2/Silva_RebekahMB_2020.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Attributes of the [4Fe4S] Cofactor Coordinated by UvrC, a DNA Repair Enzyme", "author": [ { "family_name": "Silva", "given_name": "Rebekah Miriam Brawer", "orcid": "0000-0002-9144-4939", "clpid": "Silva-Rebekah-Miriam-Brawer" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein-bound iron sulfur clusters are critical in cells and allow proteins to carry out many essential functions as electron carriers, catalysts for challenging organic reactions, and sensors of cellular environments. A wide range of protein families are known to coordinate iron sulfur clusters, and a growing category includes proteins involved in maintenance of the genome. Within the last three decades, iron sulfur clusters have been demonstrated to be important for enzymes that function in DNA repair, DNA replication, and transcription pathways. To date, iron sulfur clusters in the cubane [4Fe4S] geometry with all cysteine ligands have been exclusively reported for DNA repair and replication enzymes. In contrast to enzymes where the cofactor is necessary for active site chemistry or directly-linked to protein function, the [4Fe4S] cluster in the overwhelming majority of repair and replication enzymes is not involved in the catalytic modification of DNA substrates. Rather, the role of the cofactor appears to vary in function from protein to protein, and has been demonstrated to be important for protein stability, in the assembly of multisubunit proteins, and for substrate recognition, among other roles. Through investigations of the redox chemistry of the cofactor, our group has found that these enzymes participate in DNA-mediated charge transport chemistry, the process through which electrons rapidly migrate through well-stacked, duplex DNA. Long-range, DNA-mediated redox signaling provides a means of rapid communication among DNA-processing proteins for organizing repair and replication activities across the nucleus.
\r\n\r\nNotably, the first observations of the [4Fe4S] cofactor associated with repair and replications enzymes has consistently occurred well after the first biochemical studies of these enzymes. In some cases, the demonstration of a [4Fe4S] center has taken place decades later after initial work. Some proteins have required use of anaerobic methods in order to detect the cofactor, perhaps explaining why in some cases the metal center had eluded observation. Analysis of protein sequences might be expected to help accelerate identification of new iron sulfur centers in repair and replication enzymes. However, even with the abundance of sequencing data available in the post-genomic era, prediction of a metal center based on sequences alone has been challenging. This is in large part because the spacing of the coordinating cysteine residues can be quite irregular, leading to a weak bioinformatic signature.
\r\n\r\nIdentifying proteins with overlooked [4Fe4S] cofactors poses an exciting challenge, and there are some elegant examples in the literature where data from genetics assays has been used in combination with careful sequence analysis to predict and discover iron sulfur centers in repair and replication enzymes. Described here is the evolution of our studies on one well-known repair enzyme from Escherichia coli, UvrC. UvrC is part of the nucleotide excision repair pathway in the Bacteria domain which is responsible for addressing the wide class of bulky, helix-distorting lesions that can form after exposure to sources such as ultraviolet light, cigarette smoke, chemotherapeutics, and protein-DNA crosslinks. UvrC, an excision nuclease with two distinct active sites that incise the phosphodiester backbone on either side of the site of damage, has been historically challenging to study. Given how essential UvrC is in repairing damaged substrates, new insight has been greatly needed.
\r\n\r\nThrough integration of several key reports from the literature regarding the sequence of UvrC and evidence that pointed to a cofactor from genetics assays, our group predicted that UvrC is a [4Fe4S] protein. Development of a new overexpression system and an anaerobic purification method allowed for isolation of UvrC in holo form. We used spectroscopic techniques to confirm that the cluster type was [4Fe4S], and a combination of spectroscopy and chromatography to demonstrate that the UvrC-bound cofactor is susceptible to oxidative degradation. We also found that loss of the cofactor, either through aerobic degradation or mutation of coordinating cysteines, is associated with aggregation of apoprotein. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 \u00b1 20 nM and 80 \u00b1 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with DNA-bound midpoint potential of 90 mV vs. NHE.
\r\n\r\nThe work detailed in this dissertation has highlighted how critical the [4Fe4S] center is for UvrC, where the cofactor has been implicated in protein stabilization, substrate binding, and redox signaling on DNA. Handling an apo form of UvrC may have led to the previous challenges catalogued by researchers. Through the development of entirely new methods to study UvrC under anaerobic conditions, many opportunities are now available to study UvrC and the NER pathway anew in vitro and in vivo. Such work will contribute additional insight on how iron sulfur clusters are essential for enzymes that maintain genomic integrity.
\r\n\r\n", "doi": "10.7907/r0j6-jk09", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11282", "collection": "thesis", "collection_id": "11282", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11262018-103442842", "primary_object_url": { "basename": "AZ_thesis_FINAL.pdf", "content": "final", "filesize": 12244069, "license": "other", "mime_type": "application/pdf", "url": "/11282/1/AZ_thesis_FINAL.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Investigations of DNA-Mediated Redox Signaling Between E.coli DNA Repair Pathways", "author": [ { "family_name": "Zhou", "given_name": "Andy", "orcid": "0000-0003-3383-0855", "clpid": "Zhou-Andy" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The 4Fe4S cluster has been identified in various DNA-processing proteins spanning a variety of biological functions and all domains of life. Recently, a novel functional role for the cluster has been identified for proteins in DNA repair and replication as a redox switch for DNA binding. Human DNA primase utilizes this redox switch to coordinate primer handoff in replication. The enzymatic activity of DNA polymerase \u03b4 is tuned by the redox-switch, allowing for a fast and reversible regulation of replication in response to oxidative stress. In all cases, the redox of the 4Fe4S cluster is achieved through DNA-mediated charge transport (CT), the ability for DNA to carry charge through its \u03c0-stack. Due to the reliance of this phenomena on the \u03c0-stacking of the nitrogenous bases, DNA CT is sensitive to DNA lesions and mismatches and can proceed over long molecular distances if the DNA is well-stacked. Given this powerful biological phenomena, new inter-protein signaling interactions have been identified with important downstream consequences for genome fidelity. Here, we investigate the ways DNA-mediated charge transport between DNA processing enzymes results in efficient DNA repair or prevention of DNA-damage.
\r\n\r\nFirst, we investigated Dps, a bacterial ferritin that protects DNA from oxidative stress and implicated in bacterial survival and virulence. Dps iron sites can scavenge diffusing oxidants directly but additionally electrons and electron holes can be rapidly transported through the base-pair \u03c0-stack though DNA CT, thus providing an additional mechanism of genome protection by Dps. Using X-band EPR, we monitored formation of mononuclear high-spin Fe(III) sites of low symmetry as a gauge of effective Dps protection via oxidation of its iron sites. Using poly(dGdC)2 or poly(dAdT)2 DNA, we uncovered the dependence of DNA protection by Dps to the formation of guanine radical intermediates. Oxidation of Dps iron sites depended on the presence of the W52 residue. Point mutations of W52 revealed its involvement in an electron transfer (ET) pathway for the oxidation of the Dps iron sites. Finally, we investigated the in vivo consequences of the Dps W52 residue by complementing knockout Dps E.coli with plasmids expressing WT, W52A, or W52Y Dps and applying oxidative stress to the cells through hydrogen peroxide treatment. These assays further demonstrated the ability of Dps to protect the E.coli genome from harmful oxidants DNA-mediated electron transfer processes.
\r\n\r\nSecond, we assessed the redox properties of EndoIII and MutY, two base excision repair glycosylases containing 4Fe4S clusters, in the presence and absence of DNA. Previous work has shown these proteins to have a midpoint redox potential around 80mV vs. NHE when bound to DNA with a positive shift in potential in the absence of DNA. However, electrochemical details that define this midpoint potential have not been uncovered. Using a pyrolytic graphite edge electrode, we measured the midopoint potential of point mutations of EndoIII where point charges are flipped near the cluster (K208E, Y205H, and E200K) in the absence of DNA. Our measurements suggest that a change in a single point charge is not enough to shift the 4Fe4S cluster midpoint potential dramatically. Addition of a poly-L-glutamate polyanion introduced a slight negative shift (~20mV), but with the introduction of DNA a large negative shift was observed (70mV). Overall, binding to the DNA polyanion is the dominant effect in tuning the redox potential of the 4Fe4S cluster, helping to explain why all DNA binding proteins with 4Fe4S clusters studied to date have similar DNA-bound potentials.
\r\n\r\nWith these similar DNA-bound potentials, inter-protein redox signaling should occur. Previous works have demonstrated DNA-mediated redox signaling such as EndoIII signaling to DinG helicase, involved in R-loop maturation, increasing cellular survival by resolving deleterious R-loops. Additionally, different cluster- containing repair proteins of different functions and domains of life have been shown using atomic force microscopy (AFM) to localize to DNA mismatches through a redox switch for DNA-binding affinity. Given a DNA-mediated redox signaling system to scan the genome for lesions, the expression levels of these proteins may play a role in defining the scanning efficiency. We identified that the EndoIII E.coli knockout strain was sensitive to UV irradiation. This implies that EndoIII assists the nucleotide excision repair (NER) pathway via DNA-mediated redox signaling. However, knockout of MutY, another 4Fe4S glycosylase, does not impart the same UV sensitivity, and thus suggests key differences between MutY and EndoIII that define effective DNA-mediated redox signaling. Thus, the effect of protein expression level on the efficiency of DNA-mediated redox signaling was investigated using inducible protein expression of EndoIII to rescue UV-sensitivity. Using both plasmid-based and genome integrated constructs, we uncovered that low amounts of EndoIII expression were enough to rescue the growth defect, and overexpression of WT EndoIII leads to a greater defect caused by excess non-specific enzyme activity. These findings further informed investigation of this unique protein signaling interaction between EndoIII and NER protein UvrC.
\r\n\r\nWith proper EndoIII rescue plasmids, we further characterized the DNA- mediated redox signaling interaction between EndoIII and UvrC. Using UV-irradiation of genetic knockout strains and growth curve analysis, we demonstrate that EndoIII expression is essential for efficient repair of UV-induced DNA lesions, as measured through quantitative changes in growth lag-time when wild-type or mutant EndoIII is present in the cell. Electrochemical analysis of EndoIII point mutants quantify the DNA-CT inefficiencies that lead to the observed phenotypes. EndoIII, a BER repair protein, assists the NER pathway in the repair of UV-induced DNA lesions via DNA-mediated redox signaling. These results give evidence of a new signaling crosstalk between two distinct DNA repair pathways.
", "doi": "10.7907/G7NF-S349", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11325", "collection": "thesis", "collection_id": "11325", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12212018-140003184", "type": "thesis", "title": "Exploring the Biological Activity of Rhodium Metalloinsertors", "author": [ { "family_name": "Boyle", "given_name": "Kelsey Melinda", "orcid": "0000-0002-6728-8403", "clpid": "Boyle-Kelsey-Melinda" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Rhodium metalloinsertors are a unique family of potential anticancer agents that have been show to bind selectively to thermodynamically destabilized DNA base pair mismatches, abasic sites, and insertions/deletions (indels) in vitro. These metalloinsertors are also able to target mismatches in cells: metalloinsertors preferentially kill mismatch repair (MMR)-deficient cancer cells, which have a relative abundance of uncorrected DNA mismatches and indels, over MMR-proficient cells, which can repair these lesions. As such, these complexes have shown great promise as a potential treatment strategy for MMR-deficient cancers, which are often resistant to classic chemotherapies.
\r\n\r\nRecently, a new class of metalloinsertors that bear a rhodium-oxygen bond was synthesized and shown to have remarkable potency and selectivity towards MMR-deficient cells. We have discovered many key differences between first generation metalloinsertors and these new Rh-O metalloinsertors: (1) the MMR-selectivity of first generation metalloinsertors is heavily influenced by ancillary ligand bulk and lipophilicity, whereas the MMR-selectivity of Rh-O metalloinsertors is strong regardless of ancillary ligand properties, (2) first generation metalloinsertors have toxicities in the micromolar range while Rh-O metalloinsertors have toxicities in the nanomolar range, and (3) first generation metalloinsertors can only bind DNA via the \u0394-enantiomer while Rh-O metalloinsertors can bind DNA via both the \u0394- and \u039b-enantiomers. Excitingly, the improved potency and selectivity of these \"Rh-O\" metalloinsertors brings them into a realm of clinical relevance.
\r\n\r\nHere we examine the basis for the improved potency and selectivity of these new Rh-O metalloinsertors. A family of six Rh-O metalloinsertors that vary in the steric bulk and lipophilicity of an ancillary ligand was synthesized and characterized. Regardless of ancillary ligand identity, these Rh-O metalloinsertors exhibit nanomolar or low-micromolar toxicities and all preferentially target MMR-deficient cancer cells over MMR-proficient cells. Notably, the off-target accumulation of these metalloinsertors in mitochondria is very low. This cellular distribution is in stark contrast with first generation metalloinsertors in which increased ligand lipophilicity led to increased mitochondrial uptake and ultimately non-selective mitochondrial-mediated cell death. We believe robust selectivity of these complexes is retained in part due to their low off-target accumulation in the mitochondria, which is further complemented by the low dosing requirements of these potent therapeutic agents.
\r\n\r\nOur studies also suggest the high potency of these complexes may be due to a difference in DNA-binding abilities, which is supported by observed differences in which enantiomers can bind to DNA mismatches, differences in ligand buckling at physiological pH, and lipophilicity of the therapeutics, with Rh-O metalloinsertors being dramatically more lipophilic than their first generation counterparts. To better understand the structural basis for this increased potency, crystallographic experiments are underway. A first generation metalloinsertor was previously crystallized with mismatched DNA, and the structure was pivotal in identifying the DNA binding mode of metalloinsertion. Using similar methods, we are working to produce a high-resolution crystal structure of an Rh-O metalloinsertor with mismatched DNA in order to gain structural insights into the increased potency of these new complexes. A significant difference in DNA binding could result in different biological activation of proteins and overall higher potency of these Rh-O metalloinsertors.
\r\n\r\nFinally, as metalloinsertors are moved towards pre-clinical study, understanding their biological activity in diverse cell culture experiments is essential. We examined a metalloinsertor and the FDA approved chemotherapeutic agent cisplatin in 27 diverse colorectal cancer cell lines. The comparison of these drugs revealed the metalloinsertor to be on average five times more potent than cisplatin in this panel. The potency of the metalloinsertor in different cell lines spanned nearly three orders of magnitude and correlated with whole-cell uptake of rhodium. Additionally, a fluorescent metalloinsertor conjugate was used to quantify the number of lesions in DNA that could be targeted by metalloinsertion, a result that correlated well with the potency of a metalloinsertor across several cell lines, consistent with DNA mismatches as the effective biological target of the metalloinsertor.
\r\n\r\nThe experiments described within this thesis have allowed us to gain a better understanding of the biological activity of rhodium metalloinsertors. We have established that Rh-O metalloinsertors are distinct from first generation metalloinsertors, and that these new metalloinsertors can serve as highly tunable, potent, and mismatch-selective anticancer agents. Furthermore, this potency is observed across diverse cell lines and has been shown to correlate with the number of genomic DNA lesions that can be bound by metalloinsertion. The unique biological activity of these complexes makes them ideal candidates for the treatment of MMR-deficient cancers, and the potency and tunability of Rh-O metalloinsertors will allow for the development of previously unattainable diagnostic and therapeutic tools for MMR-deficiencies.
", "doi": "10.7907/1KNM-Y111", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:10739", "collection": "thesis", "collection_id": "10739", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03012018-094939210", "primary_object_url": { "basename": "PLB Thesis_complete.pdf", "content": "final", "filesize": 9981102, "license": "other", "mime_type": "application/pdf", "url": "/10739/73/PLB Thesis_complete.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Elucidating the Role of [4Fe4S] Clusters in DNA Replication and Repair Proteins", "author": [ { "family_name": "Bartels", "given_name": "Phillip Leon", "orcid": "0000-0002-9688-6592", "clpid": "Bartels-Phillip-Leon" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_chem" } ], "abstract": "[4Fe4S] clusters, redox cofactors, have been discovered in DNA processing enzymes ranging from bacterial base excision repair glycosylases to eukaryotic DNA polymerases. Bacterial repair proteins are activated toward redox activity when bound to DNA and can take advantage of DNA-mediated charge transport (DNA CT) to search the genome for lesions. DNA CT involves the rapid transport of charges through the \u03c0-stacked base pairs and is sharply attenuated in the presence of lesions, mismatches, or other stacking perturbations. Thus, [4Fe4S] repair proteins use this chemistry to rapidly redistribute to target lesions and communicate with one another over long distances.
\r\n\r\nThe general function of [4Fe4S] clusters in bacterial DNA repair has received much attention, but previous efforts have left several critical questions unanswered. First, while the redox potential of these proteins is affected by DNA binding, the relative importance of the negatively-charged DNA, the protein environment surrounding the cluster, and solvent has remained unclear. Second, the importance of [4Fe4S] clusters and DNA CT to human disease has never been directly addressed. The biological consequences of this chemistry are certainly a pressing issue, as numerous disease-relevant mutations in the human homologues of well-studied repair proteins have been recorded. Finally, the existence of [4Fe4S] clusters in eukaryotic DNA replication proteins in general, and in the B-family DNA polymerases in particular, was entirely unexpected. The function of the [4Fe4S] cluster in replication proteins was far from obvious, and the functional differences from repair proteins made them difficult to explain even in the context of CT signaling. Herein, these questions have been addressed using a combination of electrochemical, spectroscopic, and biochemical approaches.
\r\n\r\nFirst, we describe the use of pyrolytic graphite edge electrodes (PGE) and S K-edge X-ray absorption spectroscopy (XAS) to address the influence of protein environment, DNA, and solvation on the [4Fe4S] cluster redox potential in the bacterial base excision repair glycosylases endonuclease III (EndoIII) and MutY. The PGE surface is rough and favorable for protein binding; electron transfer can be further enhanced in the presence of carbon nanotubes. Electrochemical signals for EndoIII and MutY in the absence of DNA are large and reproducible, and a potential shift upon DNA binding is observed. With respect to studying proteins in the absence of DNA, the PGE electrode represents a significant advance over previously used highly-oriented pyrolytic graphite (HOPG), which is hydrophobic and difficult to prepare. To test the effect of protein environment on redox potential, a series of EndoIII point mutants were prepared in which the charge within 5 \u00c5 of the cluster was reversed or added in. None of these mutations induced a significant shift in redox potential relative to wild type, arguing that DNA electrostatics are the dominant factor in potential modulation. In parallel, XAS studies were performed on EndoIII and MutY in the presence and absence of DNA, and in the presence and absence of solvent. Ligating cysteinyl thiols and inorganic S atoms in the [4Fe4S] cluster absorb at different intensities in XAS depending on solvent environment and local electrostatics; these changes, in turn, directly correlate to redox potential. By XAS, DNA was found to induce a significant shift in absorbance, and thus potential; the removal of solvent had a smaller effect. Together, these studies provide new approaches for the study of DNA-binding [4Fe4S] proteins and reveal the critical role of DNA in tuning the redox potential.
\r\n\r\nSecond, we report on a novel mutation in human MUTYH identified from a colorectal cancer patient and confirmed to be pathological. MUTYH is responsible for repairing certain lesions induced by oxidative stress and is thus frequently implicated in cancer. This new variant, C306W, contains a mutation in one of the cysteines that ligates the [4Fe4S] cluster. Electrochemistry, activity and DNA binding assays, and spectroscopic analyses were performed for C306W alongside wild type MUTYH and two other disease-relevant mutants, Y179C and G396D, with an unaltered cluster environment. From this work, it is now clear that C306W can still bind a cluster, but it is susceptible to oxidative degradation to the [3Fe4S]+ state upon redox signaling in an aerobic environment. Consequently, enzymatic activity is very low, and DNA binding is poor. Overall, this represents the first complete characterization of the [4Fe4S] cluster in a human homologue of MutY, and the first demonstration of pathology resulting from a mutation that primarily affects the [4Fe4S] cluster.
\r\n\r\nMoving into DNA replication proteins, we report on the characterization of the [4Fe4S] cluster in yeast DNA polymerase (Pol) \u03b4, the eukaryotic lagging strand polymerase. Pol \u03b4 shows reversible electrochemical signals at a midpoint potential indistinguishable from EndoIII under the same conditions, and EPR spectroscopy confirms use of the [4Fe4S]3+/2+ couple. The electrochemical signal is attenuated on DNA containing an abasic site or a CA mismatch, confirming that Pol \u03b4 is capable of DNA-mediated signaling. Bulk electrolysis and photooxidation were used to oxidize Pol \u03b4 under anaerobic conditions, and activity assays were carried out using oxidized or untreated protein. Oxidation stalls replication activity, while electrochemical reduction of oxidized samples restores activity to untreated levels. These results thus reveal that cluster oxidation serves as a reversible switch regulating Pol \u03b4 activity, suggesting an in vivo role in responding to replication stress, especially oxidative stress. In an effort to address these possibilities, we have carried out preliminary efforts in the characterization of two potentially CT-deficient mutants, W1053A and Y1078A. Both mutants were found to be too structurally unstable to proceed with in vivo experiments, but they can serve to guide future efforts in this direction.
\r\n\r\nFinally, a strategy to examine charge transport through RPA-bound single-stranded DNA is reported. RPA is the eukaryotic single-stranded binding protein and forms a protective coat around vulnerable unwound DNA at replication forks. Given the importance of redox signaling in replication proteins, we aimed to use photooxidation experiments to determine if CT through RPA is a viable pathway; if so, this would open up a large set of long-range transfer pathways to [4Fe4S] proteins in replication. These efforts are ongoing, but the experimental strategy and initial efforts are discussed.
", "doi": "10.7907/Z9H1307G", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10354", "collection": "thesis", "collection_id": "10354", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07282017-141924517", "primary_object_url": { "basename": "TJZ Thesis Final Proofread and Corrected.pdf", "content": "final", "filesize": 7024906, "license": "other", "mime_type": "application/pdf", "url": "/10354/23/TJZ Thesis Final Proofread and Corrected.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Magnetic Field Effects and Biophysical Studies on DNA Charge Transport and Repair", "author": [ { "family_name": "Zwang", "given_name": "Theodore Joseph", "clpid": "Zwang-Theodore-Joseph" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA-mediated charge transport (DNA CT) is well established in both ground and excited state systems. Although theoretical models are still being developed, it is clear that the integrity of the extended \u03c0-stack of the aromatic heterocycles, the nucleic acid bases, plays a critical role. Electron donors and acceptors must be electronically well coupled into the \u03c0-stack, typically via intercalation. Perturbations that distort the \u03c0-stack, such as single-base mismatches, abasic sites, base lesions, and protein binding that kinks the double helix, attenuate DNA CT dramatically.
\r\n\r\nThis thesis encompasses work that first aims to understand how DNA duplex structure informs characteristics of DNA CT and then continues to develop an understanding of the role these structural features play in biological systems. To contextualize these advancements, this first chapter outlines foundational work that has shown ways that DNA structure influences its ability to conduct charge.
\r\n\r\nNext, experiments were conducted on magnetized DNA-modified electrodes to explore spin-selective electron transport through hydrated duplex DNA. These results show that the two spins migrate through duplex DNA with a different yield and that spin selectivity requires charge transport through the DNA duplex. Significantly, shifting the same duplex DNA between right-handed B- and left-handed Z-forms leads to a diode-like switch in spin selectivity; which spin moves more efficiently through the duplex depends upon the DNA helicity. With DNA, the supramolecular organization of chiral moieties, rather than the chirality of the individual monomers, determines the selectivity in spin, and thus a conformational change can switch the spin selectivity.
\r\n\r\nThis exquisite spin selectivity begged the question: how might biology take advantage of such a spin filter? Photolyase and cryptochromes both have been shown to exhibit magnetosensitive chemistry nearby a DNA binding pocket, and photolyase had previously been shown capable of DNA CT. Thus, electrochemical studies were conducted to monitor the repair of cyclobutane pyrimidine dimer lesions by E coli photolyase and truncated A Thaliana Cryptochrome 1 with an applied magnetic field. We find that the yield of dimer repair is dependent on the strength and angle of the applied magnetic field even when using magnetic fields weaker than 1 Gauss, though spin selective DNA CT is not involved. These data illustrate how cyclobutane dimer repair could be used in a biological compass that is informed by the angles of Earth\u2019s magnetic field.
\r\n\r\nNext DNA-mediated electrochemistry and atomic force microscopy studies were used to describe a role for redox active [4Fe4S] clusters in DNA-mediated charge transport signaling. DNA-modified electrochemistry shows that the [4Fe4S] cluster of DNA-bound DinG, an ATP-dependent helicase that repairs R-loops, is redox-active at cellular potentials and ATP hydrolysis increases DNA-mediated redox signaling. Atomic force microscopy experiments demonstrate that DinG and Endonuclease III, a base excision repair enzyme, cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. These data are then described using an equilibrium model which elucidates fundamental characteristics of this redox chemistry that allow DNA CT to coordinate the activities of DNA repair enzymes across the genome.
\r\n\r\nThe importance of the oxidation state of the redox-active [4Fe4S] cluster in the DNA damage detection process is then further explored. Together, these results show that the reduction and oxidation of [4Fe4S] clusters through DNA-mediated charge transport facilitates long-range signaling between [4Fe4S] repair proteins. The redox-modulated change in DNA-binding affinity regulates the ability of [4Fe4S] repair proteins to collaborate in the lesion detection process.
", "doi": "10.7907/Z9TT4P4H", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:11071", "collection": "thesis", "collection_id": "11071", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06112018-200314076", "primary_object_url": { "basename": "EOB Thesis 062018c.pdf", "content": "final", "filesize": 11150005, "license": "other", "mime_type": "application/pdf", "url": "/11071/1/EOB Thesis 062018c.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Redox Signaling in Eukaryotic DNA Replication and Repair", "author": [ { "family_name": "O'Brien", "given_name": "Elizabeth", "orcid": "0000-0003-2889-1688", "clpid": "O'Brien-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_chem" } ], "abstract": "DNA-mediated charge transport chemistry (DNA CT) offers an intriguing regulatory mechanism in biology, as it is long-range, rapid, and sensitive to mismatches and perturbations to base stacking. DNA-processing enzymes in all three domains of life moreover have been shown to contain [4Fe4S] clusters, commonly redox cofactors. Bacterial [4Fe4S] repair proteins have been shown to signal one another using long-range DNA-mediated charge transport (DNA CT), facilitating the redistribution to damaged genomic DNA in cells. The role of metabolically expensive, [4Fe4S] cluster cofactors in eukaryotic systems, however, was less clear than in prokaryotes.
\r\n\r\nHere we examine the chemical role of the [4Fe4S] cluster in eukaryotic DNA primase and the human base excision repair glycosylase, MUTYH. The primase cluster functions as a redox switch regulating DNA binding and redox signaling activity in humans and yeast. Yeast moreover require the primase redox switch for viability. Human MUTYH, a bifunctional glycosylase which repairs oxidative DNA lesions, performs DNA-mediated redox signaling, similarly to the bacterial homologue MutY. The MUTYH mutation which destabilizes the [4Fe4S] cluster during redox signaling, C306W, promotes degradation and loss of activity, associated with hereditary colorectal cancer.
\r\n\r\nTo assess the redox role of the human primase [4Fe4S] cluster, we perform anaerobic DNA electrochemistry on the [4Fe4S] domain of human primase (p58C), which independently binds DNA. On DNA-modified Au electrodes, we compare the redox activity of electrochemically oxidized and electrochemically reduced p58C. Oxidized [4Fe4S]3+ p58C is electrochemically active, and reduced [4Fe4S]2+ p58C state is redox-inert. This redox-driven switch is electrochemically reversible, and is mediated by a triad of conserved tyrosines between the DNA binding interface and [4Fe4S] cluster. Mutation of residues Y309, Y345, and Y347 to phenylalanine causes attenuation of redox switching on DNA. Single-atom mutations in the redox pathway moreover compromise initiation and truncation of primer synthesis but do not affect RNA polymerase activity. We find that primase truncation is gated by DNA CT in vitro; a single mismatch in the nascent primer abrogates truncation of primase products. As\r\nprimase is tethered to DNA polymerase \u03b1, a putative [4Fe4S] enzyme to which primase hands off the RNA-primed template, we propose that DNA-mediated signaling between primase and polymerase \u03b1 chemically regulates this handoff during the first steps of replication.
\r\n\r\nEukaryotic primase must bind both DNA and nucleotide triphosphates (NTPs) in order to convert to active form. Using DNA electrochemistry we show that p58C, and full-length DNA primase, display a robust, semi-reversible NTP-dependent signal on DNA, centered near 150mV vs. NHE. This signal is dependent on the tyrosine redox pathway. The presence of reversible redox activity at a physiological potential when primase is bound to DNA and NTPs suggests that reversible redox switching from the [4Fe4S]2+ to the [4Fe4S]3+ state is important for the activity of primase during replication.
\r\n\r\nThe cluster serves as a redox switch governing DNA binding in yeast primase, just as in human primase. Mutation of tyrosines 395 and 397 in yeast primase moreover, alters the same electron transfer chemistry as the mutation of their orthologues, Y345 and Y347, respectively, alters in human primase. Although these tyrosines are arranged differently in the yeast and human proteins, they perform the same reaction to affect the switch. The single-atom Y395F mutation causes some sensitivity to chemically induced oxidative stress in yeast, and single-residue mutation Y397L confers lethality in yeast cells. A constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases, regulates the affinity for RNA-primed DNA template, and is required for primase function in vivo.
\r\n\r\nWe finally characterize a novel mutation in the [4Fe4S] human base excision repair protein, MUTYH, which destabilizes the cluster environment and has pathogenic consequences. The MUTYH C306W mutation alters one of the cysteines coordinating the cluster to tryptophan. This mutation moreover is associated with hereditary colorectal cancer and causes defective DNA binding and enzymatic activity. We perform DNA electrochemistry on WT MUTYH, as well as C306W and two cancer-associated mutants, Y197C and G396D, which have an unaltered cluster environment. MUTYH variants participate in redox signaling, but C306W is destabilized upon oxidation from the [4Fe4S]2+ to the [4Fe4S]3+ state during signaling on DNA, leading to degradation to a [3Fe4S]+ cluster and loss of DNA binding and activity. A [4Fe4S] human DNA repair enzyme performs redox signaling on DNA; dysregulation of this signaling activity is linked to tumorigenesis.
\r\n", "doi": "10.7907/KGCP-SD98", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10329", "collection": "thesis", "collection_id": "10329", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06092017-062335915", "type": "thesis", "title": "Targeting DNA Mismatches with Luminescent Ruthenium Complexes", "author": [ { "family_name": "Boynton", "given_name": "Adam Nathaniel", "clpid": "Boynton-Adam-Nathaniel" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA base pair mismatches occur naturally in cells, typically as a result of errors during replication. Cells have evolved a DNA damage response pathway called mismatch repair (MMR) that identifies and corrects base pair mismatches in newly synthesized DNA. However, proteins involved in MMR can undergo mutations, rendering them incapable of correcting mismatches. Such deficiencies in MMR leads to an increase in genetic mutations and are associated with several forms of cancer. Because a higher mismatch frequency serves as an early indicator of cancer progression, DNA mismatches are a promising target in the design of small molecule therapeutics and diagnostics. In this context, transition metal complexes are prime candidates, owing to their\u00a0valuable spectroscopic and photophysical properties and versatile coordination sphere geometries. Our laboratory focuses on generating octahedral rhodium and ruthenium complexes that selectively target DNA mismatches. A class of rhodium complexes bearing sterically expansive planar ligands bind DNA mismatches with high selectivity and exhibit preferential cytotoxicity towards MMR-deficient cancer cells. These compounds bind to DNA through metalloinsertion, in which the bulky ligand inserts into the duplex at the thermodynamically destabilized mismatch site, displacing the mismatched bases into the DNA groove.
\r\n\r\nHerein we describe recent advances in the development of luminescent ruthenium complexes that selectively probe DNA mismatches. We demonstrate that [Ru(Me4phen)2(dppz)]2+ (Me4phen = 3,4,7,8-tetramethyl-1,10-phenanthroline; dppz = dipyrido[3,2-a:2\u2019,3\u2019-c]phenazine) is a DNA \u201clight switch\u201d that exhibits a significantly brighter steady-state emission in the presence of a DNA duplex containing a mismatch relative to completely well-matched DNA. Importantly, the bulky Me4phen ancillary ligands discourage deep intercalation of dppz between well-matched base pairs, and instead, [Ru(Me4phen)2(dppz)]2+ favors metalloinsertion at thermodynamically destabilized mismatches. [Ru(Me4phen)2(dppz)]2+ possesses a higher binding affinity towards a DNA mismatch relative to well-matched base pairs, and furthermore exhibits a longer excited-state emission lifetime when bound to a mismatch compared to that when intercalated at well-matched sites; both of these observations contribute to the dramatic steady-state emission enhancement detected with the mismatched DNA duplex. Additionally, we reveal that the right-handed delta (\u2206) isomer of [Ru(Me4phen)2(dppz)]2+ is the enantiomer which imparts all mismatch selectivity, consistent with the handedness of B-form DNA.
\r\n\r\nAnother mismatch-specific luminescent probe presented in this work is [Ru(bpy)2(BNIQ)]2+ (bpy = 2,2\u2019-bipyridine; BNIQ = benzo[c][1,7]naphthyridine-1-isoquinoline). In contrast to [Ru(Me4phen)2(dppz)]2+, the BNIQ complex exploits a bulky inserting ligand that selectively undergoes metalloinsertion at a DNA mismatch. This compound too exhibits a brighter steady-state emission in the presence of a mismatched duplex compared to entirely well-matched DNA, which we attribute to the fact that [Ru(bpy)2(BNIQ)]2+ possesses nearly a 500-fold higher binding affinity for the mismatch site compared to well-matched base pairs. Taken together, [Ru(Me4phen)2(dppz)]2+ and [Ru(bpy)2(BNIQ)]2+ represent two different yet valid approaches in the rational design of mismatch-specific small molecules, one based on ancillary ligand functionalization and the other on incorporating a sterically expansive inserting ligand.
\r\n\r\nA third approach towards the design of mismatch-specific luminescent ruthenium probes that is briefly explored here is the modification of the intercalating dppz ligand of [Ru(bpy)2(dppz)]2+. Bearing a dppz ligand substituted with four methyl groups, [Ru(bpy)2(tmdppz)]2+ (tmdppz = 3,4,7,8-tetramethyl dipyridophenazine) shows no luminescence discrimination between mismatched and well-matched duplexes. This observation ostensibly arises from the fact that the appended methyl groups shield the dppz phenazine nitrogen atoms from interactions with water when intercalated within the DNA.
\r\n\r\nWith mismatch-specific luminescent metalloinsertors such as [Ru(Me4phen)2(dppz)]2+ in hand, we have commenced biological investigations to see whether these compounds can serve as luminescent proxies for rhodium metalloinsertors in MMR-deficient cancer cells. Confocal microscopy of HCT116N and HCT116O cells reveals that [Ru(Me4phen)2(dppz)]2+ does preferentially localize to mitochondria, unlike potent cell-selective rhodium complexes such as [Rh(chrysi)(phen)(PPO)]2+ (PPO = 2-(pyridine-2-yl)propan-2-ol; chrysi = 5,6-chrysenequinone diimine); however, [Ru(Me4phen)2(dppz)]2+ shows some degree of nuclear entry. Here our goal is the application of the mismatch-specific luminescent probe in co-localization experiments to investigate what proteins are involved in the DNA damage response that is activated upon metalloinsertor binding in cellulo.
\r\n\r\nThe work presented here expands beyond the study of luminescent ruthenium complexes. Amino acid conjugates of the earlier-generation rhodium metalloinsertor [Rh(HDPA)2(chrysi)]3+ (HDPA = 2,2\u2019-dipyridylamine) were synthesized. While these conjugates exhibit mismatch binding affinities comparable to other rhodium metalloinsertors, they lose cell-selective biological activity, which may arise from altered uptake and/or sub-cellular localization. Finally, preliminary investigations were conducted on [Re(CO)3(pyOEt)(dppn)]+ (pyOEt = ethyl 3-(pyridin-4-yl)propanoate; dppn = benzodipyridophenazine) and [Ru(CN)(tpy)(dppz)]+ (tpy = terpyridine; CN = cyano), which were designed as IR-active probes to study the kinetics of DNA-mediated charge transport (CT) by time-resolved infrared (TRIR) spectroscopy. While these complexes do not possess the desired spectral TRIR properties as originally intended, steady-state luminescence experiments do suggest that this donor-acceptor pair is capable of undergoing DNA-mediated electron transfer.
\r\n\r\nAltogether, this work demonstrates the versatility of transition metal complexes as non-covalent probes for DNA. Importantly, through the rational modification of their three-dimensional ligand scaffold, one can achieve site-specific recognition of clinically relevant biomarkers such as DNA mismatches.
", "doi": "10.7907/Z9CF9N5M", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9063", "collection": "thesis", "collection_id": "9063", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07192015-214603085", "primary_object_url": { "basename": "DNA CT signaling within the cell_MAG_2015.pdf", "content": "final", "filesize": 4584274, "license": "other", "mime_type": "application/pdf", "url": "/9063/1/DNA CT signaling within the cell_MAG_2015.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport Signaling Within the Cell", "author": [ { "family_name": "Grodick", "given_name": "Michael Andrew", "clpid": "Grodick-Michael-Andrew" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA possesses the curious ability to conduct charge longitudinally through the \u03c0-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.
\r\n\r\nHerein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.
\r\n\r\nIn separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?
\r\n\r\nUvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.
\r\n\r\n\r\n", "doi": "10.7907/Z9F769GX", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8967", "collection": "thesis", "collection_id": "8967", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022015-103853129", "primary_object_url": { "basename": "ALFurstThesisCompiled.pdf", "content": "final", "filesize": 25945747, "license": "other", "mime_type": "application/pdf", "url": "/8967/1/ALFurstThesisCompiled.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport Devices for Protein Detection", "author": [ { "family_name": "Furst", "given_name": "Ariel Lesa", "orcid": "0000-0001-9583-9703", "clpid": "Furst-Ariel-Lesa" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the \u03c0-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.
\r\n\r\nFirst, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.
\r\n\r\nUsing low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.
\r\n\r\nFor effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 \u03bcL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.
\r\n\r\nWith the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.
\r\n\r\nAs differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.
\r\n", "doi": "10.7907/Z9KH0K88", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:9007", "collection": "thesis", "collection_id": "9007", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062015-192618908", "primary_object_url": { "basename": "Anna Arnold_2015_Thesis full version.pdf", "content": "final", "filesize": 35527821, "license": "other", "mime_type": "application/pdf", "url": "/9007/143/Anna Arnold_2015_Thesis full version.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Investigations of DNA-Mediated Protein Oxidation", "author": [ { "family_name": "Arnold", "given_name": "Anna Ruth", "clpid": "Arnold-Anna-Ruth" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA charge transport (CT) involves the efficient transfer of electrons or electron holes through the DNA π-stack over long molecular distances of at least 100 base-pairs. Despite this shallow distance dependence, DNA CT is sensitive to mismatches or lesions that disrupt π-stacking and is critically dependent on proper electronic coupling of the donor and acceptor moieties into the base stack. Favorable DNA CT is very rapid, occurring on the picosecond timescale. Because of this speed, electron holes equilibrate along the DNA π-stack, forming a characteristic pattern of DNA damage at low oxidation potential guanine multiplets. Furthermore, DNA CT may be used in a biological context. DNA processing enzymes with 4Fe4S clusters can perform DNA-mediated electron transfer (ET) self-exchange reactions with other 4Fe4S cluster proteins, even if the proteins are quite dissimilar, as long as the DNA-bound [4Fe4S]3+/2+ redox potentials are conserved. This mechanism would allow low copy number DNA repair proteins to find their lesions efficiently within the cell. DNA CT may also be used biologically for the long-range, selective activation of redox-active transcription factors. Within this work, we pursue other proteins that may utilize DNA CT within the cell and further elucidate aspects of the DNA-mediated ET self-exchange reaction of 4Fe4S cluster proteins.
\r\n\r\nDps proteins, bacterial mini-ferritins that protect DNA from oxidative stress, are implicated in the survival and virulence of pathogenic bacteria. One aspect of their protection involves ferroxidase activity, whereby ferrous iron is bound and oxidized selectively by hydrogen peroxide, thereby preventing formation of damaging hydroxyl radicals via Fenton chemistry. Understanding the specific mechanism by which Dps proteins protect the bacterial genome could inform the development of new antibiotics. We investigate whether DNA-binding E. coli Dps can utilize DNA CT to protect the genome from a distance. An intercalating ruthenium photooxidant was employed to generate oxidative DNA damage via the flash-quench technique, which localizes to a low potential guanine triplet. We find that Dps loaded with ferrous iron, in contrast to Apo-Dps and ferric iron-loaded Dps which lack available reducing equivalents, significantly attenuates the yield of oxidative DNA damage at the guanine triplet. These data demonstrate that ferrous iron-loaded Dps is selectively oxidized to fill guanine radical holes, thereby restoring the integrity of the DNA. Luminescence studies indicate no direct interaction between the ruthenium photooxidant and Dps, supporting the DNA-mediated oxidation of ferrous iron-loaded Dps. Thus DNA CT may be a mechanism by which Dps efficiently protects the genome of pathogenic bacteria from a distance.
\r\n\r\nFurther work focused on spectroscopic characterization of the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation via the flash-quench technique. Upon irradiation with poly(dGdC)2, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)2 is substituted with poly(dAdT)2, the yield of Dps oxidation is decreased significantly, indicating that guanine radicals facilitate Dps oxidation. The more favorable oxidation of Dps by guanine radicals supports the feasibility of a long-distance protection mechanism via DNA CT where Dps is oxidized to fill guanine radical holes in the bacterial genome produced by reactive oxygen species.
\r\n\r\nWe have also explored possible electron transfer intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the ferroxidase site (W52 in E. coli Dps). In comparison to WT Dps, in EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, W52Y and W52A mutants were deficient in forming the characteristic EPR signal at g = 4.3, with a larger deficiency for W52A compared to W52Y. In addition to EPR, we also probed the role of W52 Dps in cells using a hydrogen peroxide survival assay. Bacteria containing W52Y Dps survived the hydrogen peroxide challenge more similarly to those containing WT Dps, whereas cells with W52A Dps died off as quickly as cells without Dps. Overall, these results suggest the possibility of W52 as a CT hopping intermediate.
\r\n\r\nDNA-modified electrodes have become an essential tool for the study of the redox chemistry of DNA processing enzymes with 4Fe4S clusters. In many cases, it is necessary to investigate different complex samples and substrates in parallel in order to elucidate this chemistry. Therefore, we optimized and characterized a multiplexed electrochemical platform with the 4Fe4S cluster base excision repair glycosylase Endonuclease III (EndoIII). Closely packed DNA films, where the protein has limited surface accessibility, produce EndoIII electrochemical signals sensitive to an intervening mismatch, indicating a DNA-mediated process. Multiplexed analysis allowed more robust characterization of the CT-deficient Y82A EndoIII mutant, as well as comparison of a new family of mutations altering the electrostatics surrounding the 4Fe4S cluster in an effort to shift the reduction potential of the cluster. While little change in the DNA-bound midpoint potential was found for this family of mutants, likely indicating the dominant effect of DNA-binding on establishing the protein redox potential, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we proposed that the electron transfer pathway in EndoIII can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.
\r\n\r\nWhile the 4Fe4S cluster of EndoIII is relatively insensitive to oxidation and reduction in solution, we have found that upon DNA binding, the reduction potential of the [4Fe4S]3+/2+ couple shifts negatively by approximately 200 mV, bringing this couple into a physiologically relevant range. Demonstrated using electrochemistry experiments in the presence and absence of DNA, these studies do not provide direct molecular evidence for the species being observed. Sulfur K-edge X-ray absorbance spectroscopy (XAS) can be used to probe directly the covalency of iron-sulfur clusters, which is correlated to their reduction potential. We have shown that the Fe-S covalency of the 4Fe4S cluster of EndoIII increases upon DNA binding, stabilizing the oxidized [4Fe4S]3+ cluster, consistent with a negative shift in reduction potential. The 7% increase in Fe-S covalency corresponds to an approximately 150 mV shift, remarkably similar to DNA electrochemistry results. Therefore we have obtained direct molecular evidence for the shift in 4Fe4S reduction potential of EndoIII upon DNA binding, supporting the feasibility of our model whereby these proteins can utilize DNA CT to cooperate in order to efficiently find DNA lesions inside cells.
\r\n\r\nIn conclusion, in this work we have explored the biological applications of DNA CT. We discovered that the DNA-binding bacterial ferritin Dps can protect the bacterial genome from a distance via DNA CT, perhaps contributing to pathogen survival and virulence. Furthermore, we optimized a multiplexed electrochemical platform for the study of the redox chemistry of DNA-bound 4Fe4S cluster proteins. Finally, we have used sulfur K-edge XAS to obtain direct molecular evidence for the negative shift in 4Fe4S cluster reduction potential of EndoIII upon DNA binding. These studies contribute to the understanding of DNA-mediated protein oxidation within cells.
", "doi": "10.7907/Z9ZW1HVN", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8968", "collection": "thesis", "collection_id": "8968", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022015-104348919", "primary_object_url": { "basename": "AGWeidmannThesisCompiled.pdf", "content": "final", "filesize": 47094104, "license": "other", "mime_type": "application/pdf", "url": "/8968/1/AGWeidmannThesisCompiled.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates", "author": [ { "family_name": "Weidmann", "given_name": "Alyson Gloria", "orcid": "0000-0003-3876-2847", "clpid": "Weidmann-Alyson-Gloria" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.
\r\n\r\nHere, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.
\r\n\r\nWe have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.
\r\n\r\nWe further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (\u03b3H2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.
", "doi": "10.7907/Z9RX991X", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8769", "collection": "thesis", "collection_id": "8769", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02122015-130640999", "type": "thesis", "title": "DNA-Mediated Oxidation of Transcription Factor p53", "author": [ { "family_name": "Schaefer", "given_name": "Kathryn Nicole", "orcid": "0000-0003-0908-3191", "clpid": "Schaefer-Kathryn-Nicole" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the \u03c0-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.
\r\n\r\nTo determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]3+, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.
\r\n\r\nThe chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were 13C2D2-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward 13C2D2-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.
\r\n", "doi": "10.7907/Z9BV7DJQ", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:7951", "collection": "thesis", "collection_id": "7951", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09102013-094001578", "primary_object_url": { "basename": "mui_timothy_2014_thesis.pdf", "content": "final", "filesize": 24873791, "license": "other", "mime_type": "application/pdf", "url": "/7951/1/mui_timothy_2014_thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Expanding the Repertoire of DNA-Mediated Signaling in DNA Repair", "author": [ { "family_name": "Mui", "given_name": "Timothy Paul", "clpid": "Mui-Timothy-Paul" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA damage is extremely detrimental to the cell and must be repaired to protect the genome. DNA is capable of conducting charge through the overlapping \u03c0-orbitals of stacked bases; this phenomenon is extremely sensitive to the integrity of the \u03c0-stack, as perturbations attenuate DNA charge transport (CT). Based on the E. coli base excision repair (BER) proteins EndoIII and MutY, it has recently been proposed that redox-active proteins containing metal clusters can utilize DNA CT to signal one another to locate sites of DNA damage.
\r\n\t\r\nTo expand our repertoire of proteins that utilize DNA-mediated signaling, we measured the DNA-bound redox potential of the nucleotide excision repair (NER) helicase XPD from Sulfolobus acidocaldarius. A midpoint potential of 82 mV versus NHE was observed, resembling that of the previously reported BER proteins. The redox signal increases in intensity with ATP hydrolysis in only the WT protein and mutants that maintain ATPase activity and not for ATPase-deficient mutants. The signal increase correlates directly with ATP activity, suggesting that DNA-mediated signaling may play a general role in protein signaling. Several mutations in human XPD that lead to XP-related diseases have been identified; using SaXPD, we explored how these mutations, which are conserved in the thermophile, affect protein electrochemistry.
\r\n\r\nTo further understand the electrochemical signaling of XPD, we studied the yeast S. cerevisiae Rad3 protein. ScRad3 mutants were incubated on a DNA-modified electrode and exhibited a similar redox potential to SaXPD. We developed a haploid strain of S. cerevisiae that allowed for easy manipulation of Rad3. In a survival assay, the ATPase- and helicase-deficient mutants show little survival, while the two disease-related mutants exhibit survival similar to WT. When both a WT and G47R (ATPase/helicase deficient) strain were challenged with different DNA damaging agents, both exhibited comparable survival in the presence of hydroxyurea, while with methyl methanesulfonate and camptothecin, the G47R strain exhibits a significant change in growth, suggesting that Rad3 is involved in repairing damage beyond traditional NER substrates. Together, these data expand our understanding of redox-active proteins at the interface of DNA repair.
\r\n", "doi": "10.7907/WXQM-CJ32", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8412", "collection": "thesis", "collection_id": "8412", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282014-143107089", "primary_object_url": { "basename": "thesis_final.pdf", "content": "final", "filesize": 8258010, "license": "other", "mime_type": "application/pdf", "url": "/8412/1/thesis_final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Design, Synthesis, and Biological Activity of Rhodium Metalloinsertors", "author": [ { "family_name": "Komor", "given_name": "Alexis Christine", "clpid": "Komor-Alexis-Christine" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells.
\r\n \r\nHere we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle and induction of necrosis, which occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents.
\r\n\r\nIn addition, ten distinct metalloinsertors with varying lipophilicities are synthesized and their mismatch binding affinities and biological activities studied. While they are found to have similar binding affinities, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show that all of these metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. Furthermore, metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cytotoxic and antiproliferative activities that are selective for cells deficient in MMR.
\r\n\r\nTo explore further the basis of the unique selectivity of the metlloinsertors in targeting MMR-deficient cells, experiments were conducted using engineered NCI-H23 lung adenocarcinoma cells that contain a doxycycline-inducible shRNA which suppresses the expression of the MMR gene MLH1. Here we use this new cell line to further validate rhodium metalloinsertors as compounds capable of differentially inhibiting the proliferation of MMR-deficient cancer cells over isogenic MMR-proficient cells. General DNA damaging agents, such as cisplatin and etoposide, in contrast, are less effective in the induced cell line defective in MMR.
\r\n\r\nFinally, we describe a new subclass of metalloinsertors with enhanced potency and selectivity, in which the complexes show Rh-O coordination. In particular, it has been found that both \u0394 and \u039b enantiomers of [Rh(chrysi)(phen)(DPE)]2+ bind to DNA with similar affinities, suggesting a possible different binding conformation than previous metalloinsertors. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than the FDA-approved anticancer drugs cisplatin and MNNG. Moreover, these activities are coupled with high levels of selectivity for MMR-deficient cells.
\r\n", "doi": "10.7907/KPCY-JS09", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7960", "collection": "thesis", "collection_id": "7960", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09192013-114842407", "primary_object_url": { "basename": "Pheeney_Thesis Complied 09-19-2013.pdf", "content": "final", "filesize": 4478398, "license": "other", "mime_type": "application/pdf", "url": "/7960/1/Pheeney_Thesis Complied 09-19-2013.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Multiplexed DNA-Mediated Electrochemistry", "author": [ { "family_name": "Pheeney", "given_name": "Catrina Gale", "clpid": "Pheeney-Catrina-Gale" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The aromatic core of double helical DNA possesses the unique and remarkable ability to form a conduit for electrons to travel over exceptionally long molecular distances. This core of \u03c0-stacked nucleobases creates an efficient pathway for charge transfer to proceed that is exquisitely sensitive to even subtle perturbations. Ground state electrochemistry of DNA-modified electrodes has been one of the major techniques used both to investigate and to harness the property of DNA-mediated charge transfer. DNA-modified electrodes have been an essential tool for both gaining insights into the fundamental properties of DNA and, due to the exquisite specificity of DNA-mediated charge transfer for the integrity of the \u03c0-stack, for use in next generation diagnostic sensing. Here, multiplexed DNA-modified electrodes are used to (i) gain new insights on the electrochemical coupling of metalloproteins to the DNA \u03c0-stack with relevance to the fundaments of in vivo DNA-mediated charge transfer and (ii) enhance the overall sensitivity of DNA-mediated reduction for use in the detection of low abundance diagnostic targets.
\r\n\r\nFirst, Methylene Blue (MB\u2032) was covalently attached to DNA through a flexible C12 alkyl linker to yield a new redox reporter for DNA electrochemistry measurements with enhanced sensitivity. Tethered, intercalated MB\u2032 was reduced through DNA-mediated charge transport. The redox signal intensity for MB\u2032-dT-C12-DNA was found to be at least 3 fold larger than that of previously used Nile Blue (NB)-dT-DNA, which is coupled to the base stack via direct conjugation. The signal attenuation, due to an intervening mismatch, and therefore the degree of DNA-mediated reduction, does, however, depend on the DNA film morphology and the backfilling agent used to passivate the surface. These results highlight two possible mechanisms for the reduction of MB\u2032 on the DNA-modified electrode that are distinguishable by their kinetics: reduction mediated by the DNA base pair stack and direct surface reduction of MB\u2032 at the electrode. The extent of direct reduction at the surface can be minimized by overall DNA assembly conditions.
\r\n\r\nNext, a series of intercalation-based DNA-mediated electrochemical reporters were developed, using a flexible alkane linkage to validate and explore their DNA-mediated reduction. The general mechanism for the reduction of distally bound redox active species, covalently tethered to DNA through flexible alkyl linkages, was established to be an intraduplex DNA-mediated pathway. MB, NB, and anthraquinone were covalently tethered to DNA with three different covalent linkages. The extent of electronic coupling of the reporter was shown to correlate with the DNA binding affinity of the redox active species, supporting an intercalative mechanism. These electrochemical signals were shown to be exceptionally sensitive to a single intervening \u03c0-stack perturbation, an AC mismatch, in a densely packed DNA monolayer, which further supports that the reduction is DNA-mediated. Finally, this DNA-mediated reduction of MB occurs primarily via intra- rather than inter duplex intercalation, as probed through varying the proximity and integrity of the neighboring duplex DNA.\r\nFurther gains to electrochemical sensitivity of our DNA-modified devices were then achieved through the application of electrocatalytic signal amplification using these solvent accessible intercalative reporters, MB-dT-C8, and hemoglobin as a novel electron sink. Electrocatalysis offers an excellent means of electrochemical signal amplification, yet in DNA based sensors, its application has been limited due to strict assembly conditions. We describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low density DNA films. Electrocatalysis of MB that is covalently tethered to the DNA by a flexible alkyl linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of single CA mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: it is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling.
\r\n\r\nFinally, we expanded the application of our multiplexed DNA-modified electrodes to the electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins that contain redox cofactors. Multiplexed analysis of EndonucleaseIII (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, elucidated subtle differences in the electrochemical behavior as a function of DNA morphology. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction. Closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. The electrochemical comparison of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster, was able to be quantitatively performed. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. Based on the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues, but also through changes in solvation near the cluster.
\r\n", "doi": "10.7907/PCF7-9669", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7446", "collection": "thesis", "collection_id": "7446", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01282013-083506263", "primary_object_url": { "basename": "Muren_Natalie_2013_combined_chapters.pdf", "content": "final", "filesize": 5695199, "license": "other", "mime_type": "application/pdf", "url": "/7446/1/Muren_Natalie_2013_combined_chapters.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport for Long-Range Sensing and Protein Detection", "author": [ { "family_name": "Muren", "given_name": "Natalie Bloom", "clpid": "Muren-Natalie-Bloom" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The structural core of DNA, a continuous stack of aromatic heterocycles\u2014the base pairs\u2014that extends down the helical axis, gives rise to the fascinating electronic properties of this molecule that is so critical for life. This \u03c0-stacked structure facilitates a unique form of charge conduction, termed DNA-mediated charge transport (DNA CT). Experiments with diverse platforms, in solution, on surfaces, and with single molecules, collectively provide a broad and consistent perspective on the essential characteristics of this chemistry. Notably, DNA CT can proceed over long molecular distances, but is remarkably sensitive to perturbations in base pair stacking. These characteristics suggest that DNA CT may be used for long-range sensing both in nature and in nanoelectronic applications. Here, measurements of DNA CT with surface and single molecule platforms are used to (i) determine how ground state DNA CT varies over regimes of increasing distance and (ii) apply this chemistry to the electrical detection of DNA-binding proteins.
\r\n\r\nFirst, the design and fabrication of multiplexed, DNA-modified electrodes on silicon chips is reported. These lithographically patterned chips with 16 individually addressable gold electrodes allow for the measurement of DNA CT with four different types of DNA, side by side on the same surface, with four-fold redundancy. Discrimination of DNA with a single base mismatch and detection of sequence-specific restriction enzyme activity are both achieved with these chips. Scaling of these devices to microelectrode dimensions is also demonstrated. Importantly, these chips show greater reproducibility and consistency than commercially available rod electrodes. This greater signal quality, combined with the capacity to examine different samples side by side, opens the door for more complex applications of this platform.
\r\n\r\nThe fully developed, multiplexed chips are first used to compare DNA CT over short and long distance regimes. DNA is evaluated in this context because the efficacy of a long-range sensor, in either nature or nanoelectronics, is determined largely by its capacity to facilitate CT in a manner that is minimally affected by the CT distance. DNA CT over 34 nm in 100-mer monolayers is found to yield electrochemical signals that are comparable in size to shorter 17-mer DNA. Signal attenuation from a single base-pair mismatch in the 100-mer is also comparable to that for 17-mers, and confirms that CT in these 100-mer films is DNA-mediated. Efficient cleavage by a restriction enzyme indicates that the 100-mer DNA adopts a native, upright conformation. The alkanethiol linker used to anchor the DNA to the electrode is found to limit the electron-transfer rate for both DNA lengths. Thus the impact of increasing the CT distance on DNA CT is too small to be resolved by this platform, even over 34 nm. These measurements put DNA among the longest and most conductive molecular wires reported to date.
\r\n\r\nNext, DNA CT with multiplexed chips is extended to the electrochemical detection of methyltransferases, proteins that are attractive targets because of their prominent role in the initial stages of many types of cancer. Electrochemical detection of binding and activity by these proteins is achieved by two different methods. First, DNA-binding and base-flipping by these proteins disrupts the DNA \u03c0-stack and may be used for direct \u201csignal OFF\u201d detection. Using this method, the concentration- and cofactor- dependence of SssI methyltransferase, the bacterial analog of human methyltransferases, are examined. Second, methylation-conferred protection of DNA against cutting by a restriction enzyme may be used for \u201csignal ON\u201d detection of methyltransferase activity. With this approach, the use of both unmethylated and hemimethylated DNA substrates is demonstrated for the sensitive detection of both bacterial (SssI) and human (Dnmt1) methyltransferase activity. Importantly, the electrochemical format of these assays requires minimal equipment, is low cost, and may be easily applied to high throughput studies, making it an accessible option for a variety of research and clinical settings.
\r\n\r\nAlongside work with this surface, electrochemical platform, a single molecule, carbon nanotube-DNA (CNT-DNA) platform is also used to evaluate DNA CT over increasing distances and to detect protein binding. CNT-DNA devices consist of a single molecule of DNA that is made to bridge a gap cut in a CNT covalently, such that current flow through the device is DNA-mediated. Upon introduction of DNA bridges of varying length (15-mer, 60-mer, and 100-mer), the device resistance is minimally affected, echoing the result of long distance electrochemistry experiments. These devices are also used to detect SssI methyltransferase binding by the direct \u201csignal OFF\u201d method used with multiplexed chips; DNA-binding and base-flipping disrupts DNA CT and shuts off current flow through the device. CNT-DNA devices are used to electronically measure the sequence-specific, cofactor-dependent, and reversible binding of SssI. DNA methylation catalyzed by SssI is also detected based on its alteration of the protein-binding affinity of the device. This detection approach, which relies on DNA as both a recognition element and electrical transducer, represents a unique strategy for the specific, single molecule detection of protein binding and activity.
\r\n", "doi": "10.7907/6KG5-KQ87", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:6721", "collection": "thesis", "collection_id": "6721", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10282011-143721555", "primary_object_url": { "basename": "Olmon2012thesis.pdf", "content": "final", "filesize": 6459459, "license": "other", "mime_type": "application/pdf", "url": "/6721/1/Olmon2012thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Investigating DNA-Mediated Charge Transport by Time-Resolved Spectroscopy", "author": [ { "family_name": "Olmon", "given_name": "Eric Daniel", "clpid": "Olmon-Eric-Daniel" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In all organisms, oxidation threatens the integrity of the genome. Numerous studies have suggested that DNA-mediated charge transport (CT) may play an important role in the sequestration, detection, and repair of oxidative damage. To fully understand the mechanism of DNA-mediated CT, it is necessary to characterize transient intermediates that arise during the reaction and to determine the lifetimes of these intermediates. Time-resolved spectroscopy is the most appropriate experimental method for such observations. Each intermediate has a characteristic spectrum. By observing time-dependent changes in the absorption spectrum of the sample, it is therefore possible to determine what species are present at a particular time and how long it exists in solution. Experiments presented here involve the use of time-resolved spectroscopy to better understand the process of DNA-mediated CT.
\r\n\r\nThe study of DNA-mediated CT requires a robust and consistent method for triggering the CT reaction. The metal complexes that have traditionally been used for this purpose provide several advantages over organic phototriggers: they are synthetically versatile, they are stable in solution, they exhibit rich photophysics, and many are strong photooxidants. However, the spectroscopic features used to follow the photochemical processes triggered by these probes are generally broad optical bands. These can be difficult to resolve in samples that contain several absorbing species. For this reason, we have developed a Re photooxidant bearing a set of vibrationally active carbonyl ligands that can be covalently tethered to DNA. Unlike many absorption bands in the visible range, the vibrational absorption bands of these ligands are narrow, well-resolved, and specific. Such probes can be used to follow the complex photophysical pathways observed in biochemical systems with good precision, making them useful for the study of DNA-mediated CT.
\r\n\r\nSpecifically, the complex [Re(CO)3(dppz)(py′-OR)]+ (dppz = dipyrido[3,2-a:2′,3′-c]-phenazine; py′-OR = 4-functionalized pyridine) offers IR sensitivity and can oxidize DNA directly from the excited state. The behavior of several covalent and noncovalent Re-DNA constructs was monitored by time-resolved IR (TRIR) and UV/visible spectroscopies, as well as biochemical methods, confirming the ability of the complex to trigger long-range oxidation of DNA. Optical excitation of the complex leads to population of metal-to-ligand charge transfer excited states and at least two distinct intraligand charge transfer excited states. Several experimental observations are consistent with charge injection by excited Re*. These include similarity between TRIR spectra and the spectrum of reduced Re observed by spectroelectrochemistry, the appearance of a guanine radical signal in TRIR spectra, and the eventual formation of permanent guanine oxidation products. The majority of reactivity occurs on the ultrafast time scale, although processes dependent on slower conformational motions of DNA, such as the accumulation of oxidative damage at guanine, are also observed.
\r\n\r\nThe photooxidation activity of this Re complex was compared directly to that of other metallointercalators that have been used previously in our laboratory to oxidize DNA. The complexes [Rh(phi)2(bpy′)]3+ (phi = 9,10-phenanthrenequinone diimine; bpy′ = 4-methyl-4′-(butyric acid)-2,2′-bipyridine), [Ir(ppy)2(dppz′)]+ (ppy = 2-phenylpyridine; dppz′ = 6-(dipyrido[3,2-a:2′,3′-c]phenazin-11-yl)hex-5-ynoic acid), and [Re(CO)3(dppz)(py′-OH)]+ (py′-OH = 3-(pyridin-4-yl)-propanoic acid) were each covalently tethered to DNA. Biochemical studies show that upon irradiation, the three complexes oxidize guanine by long-range DNA-mediated CT with the efficiency: Rh > Re > Ir. Comparison of spectra obtained by spectroelectrochemistry after bulk reduction of the free metal complexes with those obtained by transient absorption (TA) spectroscopy of the conjugates suggests that excitation of the conjugates at 355 nm results in the formation of the reduced metal states. Electrochemical experiments and kinetic analysis of the TA decays verify that the primary factors responsible for the trend observed in the guanine oxidation yield of the three complexes are the thermodynamic driving force for CT, variations in the efficiency of back electron transfer, and coupling to DNA.
\r\n\r\nThe ability of redox-active DNA-binding proteins to act as hole sinks in DNA-mediated CT systems was also studied by time-resolved spectroscopy. Such experiments are designed to provide support for the utilization of DNA-mediated CT in biological systems. In studies involving the cell cycle regulator p53, photoexcitation results in the formation of a weak transient band at 405 nm. This band, which is not observed in samples lacking the protein, resembles the primary spectral feature of the tyrosine cation radical. Although the signal is weak and reproducibility is inconsistent, these results suggest that photolysis of the sample leads to DNA-mediated oxidation of tyrosine in p53. Similar experiments were conducted on the transcriptional activator SoxR. Here, the presence of dithionite, required in solution to keep the protein reduced, complicates the photochemistry of the system considerably. Regardless, a weak absorbance at 418 nm that develops following photolysis at 355 nm provides evidence for the DNA-mediated oxidation of the protein. The behavior of the base excision repair protein endonuclease III was also observed in the presence of DNA and metal complex oxidants. In flash-quench studies, addition of the protein results in the formation of a strong negative signal at 410 nm in TA traces. In studies involving direct photooxidation by Rh, Ir, and Re complexes, no new transients are detected upon the addition of protein, but changes in the intensities of the resultant TA spectra and in the steady-state absorbance spectra following photolysis indicate that DNA-mediated oxidation of the protein may be taking place.
\r\n\r\nThe experiments described here comprise several new developments in the story of DNA-mediated CT. First, proof of concept has been given for a valuable new vibrationally-active Re probe. Further modifications on the characteristics of this complex and further study by time-resolved vibrational spectroscopy will allow us to observe DNA-mediated CT with high spectral resolution. Second, comparison between this Re probe and established photooxidants shows that the Re complex is a strong photooxidant in its own right and that this complex can be added to our growing toolbox of CT phototriggers. Third, time-resolved studies involving redox-active proteins have provided preliminary direct evidence for the ability of these proteins to serve as CT probes themselves. Further refinement of the experimental methods used in these experiments will allow us to observe such processes with greater sensitivity, increasing our knowledge of the mechanism and applications of DNA-mediated CT.
\r\n", "doi": "10.7907/6GDE-2707", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6731", "collection": "thesis", "collection_id": "6731", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11072011-185116656", "primary_object_url": { "basename": "Song_thesis_2012.pdf", "content": "final", "filesize": 21244626, "license": "other", "mime_type": "application/pdf", "url": "/6731/1/Song_thesis_2012.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Recognition of Nucleic Acid Mismatches by Luminescent Ruthenium Complexes", "author": [ { "family_name": "Song", "given_name": "Hang", "clpid": "Song-Hang" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Deficiencies in DNA mismatch repair (MMR) have been implicated in the development of several forms of cancers, and MMR-deficient cells tend to be resistant to commonly employed cancer therapeutics such as cisplatin. Mismatch-targeting metalloinsertors developed in our laboratory have shown great promise as therapeutic and diagnostic agents for MMR-deficient cancers. In this work, we examine fundamental aspects of binding interactions of octahedral rhodium and ruthenium complexes to DNA mismatches, and strive to develop a luminescent sensor for mismatches inside cells.
\r\n\r\nWe first demonstrate that the mismatch binding affinity of rhodium metalloinsertors directly correlates with their antiproliferative effect against MMR-deficient colorectal carcinoma cells. Smaller ancillary ligands on the rhodium center facilitate binding to mismatches via metalloinsertion from the narrow minor groove of DNA. Complexes with higher mismatch binding affinity in turn selectively inhibit the growth of MMR-deficient cells compared to MMR-proficient ones. This correlation suggests that DNA mismatches are indeed the biological target of rhodium metalloinsertors inside cells.
\r\n\r\nBesides rhodium metalloinsertors, luminescent ruthenium complexes are found to bind DNA mismatches as well. Mismatch binding is accompanied by enhanced luminescence intensity. We determined two crystal structures of \u0394-Ru(bpy)2dppz2+ bound to oligonucleotide duplexes. For an oligonucleotide containing AA mismatches, the atomic-resolution structure revealed that the ruthenium complex binds to DNA mismatches also through metalloinsertion: the complex inserts a planar ligand into the mismatched site from the minor groove, ejecting the mismatched bases out of the helix. Several binding geometries of the complex intercalated between well-matched DNA were also observed.
\r\n\r\nTo improve the mismatch selectivity of luminescent ruthenium complexes, we tethered the complexes to organic dye molecules in an effort to amplify mismatch-associated luminescence signal through resonance energy transfer. We also modified the structure of the inserting ligand in an attempt to improve the binding affinity to mismatches over well-matched DNA. Coupling mismatch binding to luminescence response has proved most challenging in these endeavors.
\r\n\r\nFinally, we venture into the realm of RNA. Unlike their nonspecific binding to DNA, ruthenium complexes bind poorly to well-matched RNA but quite avidly to RNA mismatches. As a result, mismatched RNA produces a higher luminescence signal from bound ruthenium. We subsequently applied the ruthenium complex to image RNA mismatches inside live HeLa cells using fluorescence microscopy.
", "doi": "10.7907/4TM6-BR89", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:7072", "collection": "thesis", "collection_id": "7072", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05242012-110320685", "type": "thesis", "title": "DNA-mediated Charge Transport in a Biological Context: Cooperation among Metalloproteins to Find Lesions in the Genome", "author": [ { "family_name": "Sontz", "given_name": "Pamela Alisa", "clpid": "Sontz-Pamela-Alisa" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. A molecular wire, DNA conducts charge with shallow distance dependence, yet mismatches and lesions attenuate this process. We have proposed a model where repair proteins, containing redox-active [4Fe4S] clusters, utilize DNA charge transport (CT) to scan the genome for lesions. Based on this model, proteins are predicted to redistribute onto strands where DNA CT is inhibited. Using single-molecule atomic force microscopy (AFM) we have probed the redistribution of EndoIII, a base excision repair protein that contains a [4Fe4S] cluster. Consistent with the model, we find a redistribution of EndoIII onto DNA strands (3.8 kbp) containing C:A mismatch, which is not a specific substrate of EndoIII but inhibits CT. Proteins with mutations making them deficient in DNA-mediated CT do not similarly redistribute onto mismatched strands.
\r\n\r\nVarious DNA-binding proteins, such as those involved in repair and pathways that maintain the integrity of DNA, have been found to contain FeS domains and other redox cofactors. We are discovering proteins from alternate repair pathways that may also utilize DNA CT to find damage. XPD, a 5\u2032-3\u2032 helicase involved in nucleotide excision repair, contains a conserved [4Fe4S] cluster and exhibits a DNA-bound redox potential that indicates it is able to carry out DNA CT. In AFM studies, we observe also the redistribution of XPD onto strands containing a mismatch. We further demonstrate that an XPD mutant, L325V, defective in carrying out DNA CT, does not redistribute onto mismatched strands.
\r\n\r\nDNA CT between distinct repair proteins bound to DNA was also probed by AFM. When XPD and EndoIII are mixed together, they coordinate in relocalizing onto mismatched strands. However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs. These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between repair proteins in their search for damage in the genome.
\r\n", "doi": "10.7907/9YT4-7181", "publication_date": "2012-05-07", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6708", "collection": "thesis", "collection_id": "6708", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10062011-140224145", "primary_object_url": { "basename": "Thesis_final_wmg.pdf", "content": "final", "filesize": 4162574, "license": "other", "mime_type": "application/pdf", "url": "/6708/1/Thesis_final_wmg.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Regulation of Wild-Type and Mutant p53 through DNA-mediated Charge Transport", "author": [ { "family_name": "Geil", "given_name": "Wendy Mercer", "clpid": "Geil-Wendy-Mercer" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The global transcription factor p53 controls many cellular processes, including the cellular response to oxidative stress. It had been determined that that dissociation of wild type p53 from its promoter site can occur upon DNA-mediated oxidation. In this work, we use site-directed mutagenesis to construct charge-deficient mutants of p53; the chemistry of DNA-mediated oxidation of p53 was examined using these mutants.
\r\n\r\nThe control point for p53 oxidation through DNA-mediated charge transport (DNA CT) is cysteine 275. Using differential thiol labeling and detection of modified peptides with mass spectrometry, we demonstrated that cysteines 124 and 141 in superstable p53 form a terminal disulfide bond upon DNA-mediated oxidation. This leads to a conformational change that inhibits DNA from binding by p53. The disulfide formed between cysteines 124 and 141 is a result of a series of disulfide bond exchange across the protein from the DNA base stack.
\r\n\r\nWe also investigated the dependence of p53 oxidation on DNA sequences. ESMA analysis of biologically derived p53 recognition sequences with varying quantities of guanine doublets and triplets showed efficient p53 oxidation to depend on the presence of low energy GG or GGG sites. Moreover, consistent results were found with biologically derived promoter sequences. Sequence S100A2, with guanine triplets on the same strand, showed the most oxidation of p53, followed by ODC1 and caspase-1. We confirmed these sequence-specific effects by measuring the change in expression level of the genes after induction of DNA CT in vivo. S100A2 mRNA levels decreased after photooxidant and light treatment, reflecting the oxidation and dissociation of p53 from the S100A2 site. However, caspase-1 and ODC1 mRNA levels remained the same, indicating less DNA-mediated p53 oxidation.
\r\n\r\nThe results from this study illustrate how protein oxidation at a distance through DNA CT contributes to cellular signaling. This oxidative signaling can control how p53 regulates gene expression under oxidative stress, and this signaling may be disrupted in cancerous cells.
\r\n", "doi": "10.7907/0NVZ-QC23", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6277", "collection": "thesis", "collection_id": "6277", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04032011-125158842", "primary_object_url": { "basename": "Complete_Thesis.pdf", "content": "final", "filesize": 6687006, "license": "other", "mime_type": "", "url": "/6277/12/Complete_Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transfer Between [4Fe-4S] Cluster Glycosylases", "author": [ { "family_name": "Romano", "given_name": "Christine Anne", "clpid": "Romano-Christine-Anne" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The work performed herein describes three proteins: Uracil DNA glycosylase (UDG) from Archaeoglobus fulgidus, MutY, and Endonuclease III (EndoIII) from Escherichia coli. They are DNA repair glycosylases that contain [4Fe-4S] clusters. While the catalytic mechanisms of many BER enzymes have been studied in detail, questions remain about how these enzymes search the vast amount of cellular DNA to find their substrates, and why some require a [4Fe-4S] cluster. The iron-sulfur cluster is not necessary for catalysis, and it only displays a physiologically relevant midpoint potential when bound to DNA. We have proposed that UDG, MutY, and EndoIII use their [4Fe-4S] clusters to participate in DNA-mediated charge transport (CT), and that these proteins mediate long-range electrochemical signaling in order to detect DNA damage.
\r\n\r\nThis scheme for DNA damage detection assumes that CT occurs efficiently between the DNA helix and the [4Fe-4S] cluster of the bound protein. In order for efficient CT to occur, a pathway of amino acids must be present that facilitates CT between the DNA and the iron-sulfur cluster. For each of the enzymes mentioned, this pathway was explored through mutagenesis. In UDG, MutY, and EndoIII, several amino acids thought to be important for CT were mutated and the resulting proteins were characterized biochemically. Their CT capabilities were analyzed by cyclic voltammetry on DNA-modified electrodes. In these experiments, the mutants\u2019 signal intensities were quantified and compared to those of wild-type enzyme. An attenuated signal relative to wild-type protein may indicate that the mutant is deficient in CT and that the targeted amino acid is part of the protein-DNA CT pathway in the native enzyme. Many mutants were also screened by enzymatic assays and circular dichroism spectroscopy to further characterize their DNA-binding properties and structural stability.
\r\n\r\nThe A. fulgidus UDG mutants examined, C17H, C85S, and C101S, all contained mutations in the cysteine residues that ligate the [4Fe-4S] cluster. These mutants were designed to determine how the iron-sulfur cluster coordination environment affects protein-DNA CT. The mutants exhibited varying signal strengths relative to WT UDG on DNA-modified electrodes. C85S produced a weaker signal, indicating a CT deficiency. The signal intensity from C101S was within error of that of WT, and the signal from C17H was larger than that of WT, possibly indicating that this mutant is less structurally stable than WT UDG.
\r\n \r\nIn E. coli MutY, position Y82 aligns with Y165 in MUTYH, a residue in which mutations have been found in many colorectal cancer patients. To better understand the correlation between protein-DNA CT and colorectal cancer, the MutY mutants Y82C and Y82L were prepared and characterized. Y82C exhibited a CT deficiency relative to WT MutY, whereas Y82L did not. These data indicate that Y82 forms part of the CT pathway in native E. coli MutY, but that other long-chain amino acids, such as leucine, can also mediate CT efficiently at this position.
\r\n \r\nSeveral different mutants of E. coli EndoIII were examined. First, the Y82 position was targeted, since the aligning MUTYH residue has been found mutated in colorectal cancer patients and because this residue is located near the protein-DNA interface. Five mutations were made at or near the Y82 position, and their cyclic voltammetry signals demonstrated that aromatic amino acids best mediate CT at this position. Other residues towards the interior of the protein, Y75, Y55, and F30 were also mutated to alanines. These mutants exhibited CT deficiencies, implicating the residues as part of a potential CT pathway. Residues W178 and Y185, located near the [4Fe-4S] cluster of EndoIII, were also mutated to alanines. The resulting mutants produced larger signals than that of WT EndoIII. These mutants were later shown by circular dichroism spectroscopy to be less stable structurally than WT EndoIII. All of the mutants mentioned exhibited enzymatic properties similar to those of WT, suggesting that they are able to bind DNA and excise damage nucleobases as well as the native enzyme. Several of these mutants were also used in a mutagenesis-based experiment to assay how EndoIII variants help MutY search for DNA lesions, although data from these experiments showed no significant differences in mutation rate between strains expressing different EndoIII variants.
\r\n \r\nIn total, the mutagenesis studies performed here helped determine the characteristics of BER enzymes that enable them to mediate DNA-protein CT. All these enzymes must contain a stable, well-protected metallocluster that charge can access through a series of CT-facilitating amino acids. In discovering several residues important for protein-DNA CT in UDG, MutY, and EndoIII, we have strengthened support for the hypothesis that these enzymes facilitate DNA-mediated CT in vivo. These enzymes may in fact be part of a much larger array of redox-active DNA-binding proteins that communicate electrochemically to help each other detect and repair DNA lesions inside the cell.
\r\n", "doi": "10.7907/63TC-FN74", "publication_date": "2011-06-10", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6464", "collection": "thesis", "collection_id": "6464", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272011-113632785", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 10925182, "license": "other", "mime_type": "application/pdf", "url": "/6464/1/Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "The Biological Activity of Rhodium Metalloinsertors", "author": [ { "family_name": "Ernst", "given_name": "Russell J.", "clpid": "Ernst-Russell-J" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. The loss of MMR carries dire consequences, including increased mutation rates, carcinogenesis, and resistance to a variety of clinical anti-cancer agents, such as cisplatin and DNA alkylators. Rhodium metalloinsertors previously developed in our laboratory bind to DNA mismatches with high affinity and specificity, and represent a promising strategy to target mismatches in cells. Thus, uncorrected mismatches can be exploited to provide a basis of discrimination between MMR-deficient, cancerous cells and MMR-proficient, healthy cells.
\r\n\r\nHere we describe the application of rhodium metalloinsertors to inhibit cellular proliferation selectively in MMR-deficient cells compared to those that are MMR-proficient. The colorectal carcinoma cell lines HCT116N and HCT116O serve as an isogenic model system for MMR deficiency. We show that the \u0394-isomer of an octahedral rhodium complex containing a bulky chelate ligand for insertion into a DNA mismatch is active both in targeting base mismatches in vitro and in inhibiting DNA synthesis selectively in the HCT116O cell line.
\r\n\r\nA family of derivative complexes with varying ancillary ligands has also been synthesized, and both DNA mismatch binding affinities and anti-proliferative activities against the HCT116 cell lines have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatched sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M-1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo.
\r\n\r\nIn particular, rhodium metalloinsertors bearing dipyridylamine ancillary ligands are shown to exhibit accelerated cellular uptake. This increased uptake allows us to observe additional cellular responses to these agents, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that also occur preferentially in the HCT116O cell line. Finally, these cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents, and are consistent with the idea that repair proteins are activated in response to DNA mismatch binding.
\r\n", "doi": "10.7907/3K6K-JX55", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5500", "collection": "thesis", "collection_id": "5500", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01062010-125002622", "primary_object_url": { "basename": "JCGThesis.pdf", "content": "final", "filesize": 14836624, "license": "other", "mime_type": "", "url": "/5500/7/JCGThesis.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Exploring DNA-Mediated Charge Transport with Fast Radical Traps", "author": [ { "family_name": "Genereux", "given_name": "Joseph Charles", "orcid": "0000-0002-5093-7710", "clpid": "Genereux-Joseph-Charles" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" }, { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The \u03c0-stack of DNA is competent for mediating charge transport (CT), both by single-step and multi-step mechanisms. The yield of long-range single-step CT from photoexcited 2-aminopurine to guanine across adenine tracts has a shallow, periodic distance dependence, with increasing amplitude and decreasing slope with temperature. To measure total CT yield, herein we employ the fast radical traps N2-cyclopropylguanine (CPG), and N6-cyclopropyladenine (CPA), which are similar to the unmodified bases, but undergo rapid decomposition upon oxidation. We find that decomposition of CPG by a photoexcited rhodium intercalator across an adenine tract has similar periodic distance dependence to quenching of 2-aminopurine by guanine, and the same temperature dependence as well. In contrast, decomposition of CPG by photoexcited 2-aminopurine is monotonic with respect to adenine tract length, and also competes with back electron transfer (BET). Eliminating BET by separating 2-aminopurine from the adenine tract with three high-potential inosines restores the non-monotonic distance dependence. We also determined decomposition of CPA along adenine tracts by photoexcited rhodium, and found the CT yield to be distance-independent, demonstrating that the periodicity associated with guanine oxidation is with respect to adenine tract length, not donor-acceptor separation. This length-dependent periodicity, and the associated temperature dependence, support a model of conformational gating in the formation of CT-active domains along the DNA.
\r\n\r\nDNA-mediated electrochemistry is facile in self-assembled monolayers on electrodes, and redox-active dyes are reduced through the DNA \u03c0-stack at potentials far lower than those of the individual bases. Since cytosine is the most readily reduced base, we incorporated CPC into DNA monolayers to assay for bridge occupation, and CPC decomposition was not observed.
\r\n\r\nTo explore the relative contributions of single-step and multi-step mechanisms to CT yield across adenine tracts, we compared quantum yields previously collected from 2-aminopurine fluorescence quenching experiments to those of CPG decomposition. For seven or eight intervening adenines, single-step CT accounts for the entire CT yield, while for four to six adenines, multi-step CT is the dominant mechanism. We interrupted multi-step CT by substituting CPA for an adenine on the bridge, and found the total CT yield across five or six intervening adenines is lowered to the single-step CT yield. Blocking single-step CT by replacing the terminal guanine with redox-inactive inosine does not affect CPA decomposition on the bridge. These results imply that single-step and multi-step CT processes are not in direct competition for these assemblies, consistent with the model of conformationally gated CT-active states.
\r\n", "doi": "10.7907/R0CF-GF80", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5269", "collection": "thesis", "collection_id": "5269", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06162009-143222", "primary_object_url": { "basename": "FullThesis1.pdf", "content": "final", "filesize": 73920847, "license": "other", "mime_type": "application/pdf", "url": "/5269/11/FullThesis1.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Investigations into the Generality of Metalloinsertion at DNA Defects", "author": [ { "family_name": "Zeglis", "given_name": "Brian Matthew", "clpid": "Zeglis-Brian-Matthew" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Metalloinsertors are substitutionally inert, octahedral transition metal complexes that bind to thermodynamically destabilized mismatched sites in duplex DNA with high affinity and selectivity. The complexes approach DNA from the minor groove, eject the mismatched bases into the major groove, and replace the displaced bases in the helical \u03c0-stack with their own sterically expansive ligands. Herein, we describe a series of five investigations aimed at elucidating the generality of metalloinsertion at DNA defects.
\r\n\r\nIn an effort to develop a diagnostic for mismatched DNA, a bifunctional, mismatch-specific conjugate with rhodium metalloinsertor and fluorophore subunits has been constructed. A proof-of-concept conjugate was successfully produced that displays an almost fourfold fluorescence enhancement in the presence of mismatched versus matched DNA.
\r\n\r\nTo investigate the range of metal complexes capable of mismatch-specific metalloinsertion, a ruthenium bisdipyridyl complex bearing the heptacyclic eilatin ligand has been synthesized and characterized. Electrophoresis competition experiments illustrate that the complex does display mismatch-preferential, though not necessarily mismatch-selective, binding.
\r\n\r\nTo probe the generality of metalloinsertion at other common thermodynamically destabilized DNA defects, the binding of rhodium metalloinsertors at abasic sites and single base bulges has been studied. It was determined that metalloinsertors bind abasic sites with high affinity and specificity, without regard to the identity of the unpaired base and with little dependence on the sequence context of the defect. Single base bulge recognition proved more elusive, with both the identity of the unpaired base and the sequence context influencing recognition.
\r\n\r\nTo determine the structural generality of metalloinsertion, single crystal X-ray diffraction was employed to determine the structure of \u0394-Rh(bpy)2(chrysi)3+ bound to an oligonucleotide duplex containing two A\u2022A mismatches. Two structures were obtained at <2 \u00c5 resolution, and each provides an archetypical picture of metalloinsertion: the bulky rhodium complex inserts into the mismatched site from the minor groove, ejecting the mismatched bases and replacing the displaced base pair with its own sterically expansive ligand.
\r\n\r\nFinally, two mismatch-specific conjugates have been designed for chemotherapeutic applications: a metalloinsertor-oxaliplatin conjugate for the selective delivery of platinum chemotherapeutics to mismatch repair deficient cells and a metalloinsertor-Auger electron emitter conjugate for the selective irradiation of mismatch-containing DNA.
", "doi": "10.7907/0YRQ-3W54", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5618", "collection": "thesis", "collection_id": "5618", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03192010-175848794", "primary_object_url": { "basename": "PEL_Thesis_Caltech_rev2.pdf", "content": "final", "filesize": 2328398, "license": "other", "mime_type": "application/pdf", "url": "/5618/1/PEL_Thesis_Caltech_rev2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Activation of Transcription from a Distance: Investigations on the Oxidation of SoxR by DNA-Mediated Charge Transport\r ", "author": [ { "family_name": "Lee", "given_name": "Paul Eulehwann", "clpid": "Lee-Paul-Eulehwann" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In enteric bacteria, the cellular response to oxidative stress caused by superoxide is activated by soxR, which encodes a redox-active transcription factor that contains a [2Fe2S] cluster and binds DNA with high affinity. Here we describe how SoxR may detect global changes in oxidative stress while bound to DNA at a single location through DNA-mediated charge transport. A unique property of DNA is its ability to delocalize charge along its base stack, allowing oxidative damage to be funneled to specific sites of low oxidation potential. Charge transport also has the potential to access proteins with redox-active moieties.
\r\n\r\nElectrochemical studies presented here demonstrate that the redox couple of the [2Fe2S] clusters of SoxR can be accessed through the DNA, and that when the protein is bound to DNA, is shifted almost 0.5 V positive to its potential measured in solution in the absence of DNA. SoxR in its reduced form is found to inhibit guanine damage by repairing guanine radicals formed in DNA by the use of various photoactive metallointercalators, by donating an electron from one of its [2Fe2S]\u207a clusters and filling the guanine radical hole. RT-PCR is used to monitor the amount of soxS mRNA produced in cells that have taken up the DNA binding photooxidant [Rh(phi)2bpy]\u00b3\u207a and are treated with light. Cells thus treated to generate guanine radicals express soxS, evidence that SoxR is being oxidized. An in vitro assay is furthermore used to examine directly the DNA-mediated oxidation of SoxR by measuring its transcriptional activity. [Rh(phi)2bpy']\u00b3\u207a, tethered to DNA 80 bp from the soxS promoter, induces transcription by activating SoxR upon irradiation. These results demonstrate not only that guanine radicals can act to oxidize SoxR, but that the resulting oxidized, DNA-bound protein is biologically active. Thus, transcription can be activated from a distance through DNA-mediated charge transport.
\r\n\r\nThe ability of DNA to conduct charge along its base stack allows offers a general strategy for DNA-mediated signaling of oxidative stress, as it allows information about oxidative events to be transmitted quickly and directly to the proteins responsible for turning on the genes necessary for cell survival.
\r\n", "doi": "10.7907/44P4-C408", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5499", "collection": "thesis", "collection_id": "5499", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01062010-122905478", "primary_object_url": { "basename": "CAP_thesis.pdf", "content": "final", "filesize": 11296575, "license": "other", "mime_type": "application/pdf", "url": "/5499/8/CAP_thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "The Cellular Uptake of Luminescent Ruthenium Complexes", "author": [ { "family_name": "Puckett", "given_name": "Cindy Ann", "clpid": "Puckett-Cindy-Ann" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Transition metal complexes have enormous potential as diagnostic and therapeutic agents, but their internalization and distribution in living cells are only poorly understood. Here, we perform one of the few systematic explorations of the uptake efficiency and mechanism of a class of metal complexes: luminescent dipyridophenazine (dppz) complexes of ruthenium(II). Substitution of the ancillary ligands permits variation in the overall complex charge, size, and hydrophobicity. We find that internalization of these complexes occurs mostly through passive diffusion, driven by the membrane potential, and that hydrophobicity, rather than size, is the most important determinant of compound accumulation. Across different cell types with all compounds, mostly uneven cytoplasmic staining is observed with near exclusion from the nucleus. Conjugation to cell-penetrating peptides, such as D-octaarginine, increases uptake efficiency, but leads to trapping in endosomes below a threshold concentration. Above this threshold concentration, substantial staining of the nucleus as well as the cytosol is observed. An appended fluorescein tag lowers the threshold concentration, indicating the importance of payload to the internalization and distribution of cell-penetrating peptides. Shorter peptides, including the nuclear targeting signal RrRK (where r = D-arginine), are also studied, though none have as high a degree of uptake nor as low a threshold concentration as the octaarginine conjugate. These studies provide a basis for the future design and optimization of metal complexes for biological application. ", "doi": "10.7907/2484-1405", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5261", "collection": "thesis", "collection_id": "5261", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12102008-101354", "primary_object_url": { "basename": "Acknowledgements.pdf", "content": "final", "filesize": 95334, "license": "other", "mime_type": "application/pdf", "url": "/5261/1/Acknowledgements.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Electrical Detection of DNA Binding Proteins", "author": [ { "family_name": "Gorodetsky", "given_name": "Alon A.", "clpid": "Gorodetsky-Alon-A" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The base pair stack of double helical DNA has proven to be an effective medium for charge transport. The \u03c0-stacked DNA base pairs can mediate charge transport (CT) chemistry over distances as long as 20 nm, and the reaction is exquisitely sensitive to DNA sequence-dependent conformation and dynamics. This sensitivity to perturbations in DNA structure and base pair stacking makes DNA-mediated charge transport chemistry an ideal methodology for the electrical detection of base mismatches, lesions, and protein binding. Efforts toward expanding the scope of electrochemistry at DNA-modified surfaces for biosensing applications are presented here.", "doi": "10.7907/VPKN-CV38", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2408", "collection": "thesis", "collection_id": "2408", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06022008-092549", "primary_object_url": { "basename": "thesis_final_052708.pdf", "content": "final", "filesize": 18265663, "license": "other", "mime_type": "", "url": "/2408/13/thesis_final_052708.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport in DNA Repair", "author": [ { "family_name": "Boal", "given_name": "Amie Kathleen", "orcid": "0000-0002-1234-8472", "clpid": "Boal-Amie-Kathleen" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The double-helical structure of deoxyribonucleic acid (DNA) imparts upon this important biological molecule both the ability to store genetic information within a cell and also the capacity to serve as medium for charge transport. DNA-mediated charge transport is now a very well-studied phenomenon but biological roles for these reactions have not been explored. It has been demonstrated that DNA-mediated charge transport can funnel oxidative DNA damage to sites of low oxidation potential in a number of biologically relevant environments ranging from reconstituted nucleosome core particles, to isolated nuclei and mitochondria from HeLa cells. DNA-mediated charge transport may also play a role in transcriptional activation or repression as modulated by redox-active transcription factors. Here we examine how DNA-mediated charge migration could also provide a pathway for protein-protein communication among DNA repair enzymes, a pathway that might serve as a scheme for rapid lesion detection inside the cell.", "doi": "10.7907/CMWB-6C92", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2763", "collection": "thesis", "collection_id": "2763", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06282007-105808", "type": "thesis", "title": "DNA-Mediated Hole and Electron Transport", "author": [ { "family_name": "Shao", "given_name": "Fangwei", "orcid": "0000-0003-2007-3920", "clpid": "Shao-Fangwei" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Since the elucidation of the double helical structure of DNA, it has been proposed that the dynamic [pi]-stacking base pair array may mediate charge migration, hole transport (HT), and electron transport (ET). In this thesis work, both DNA-mediated HT and ET are investigated to explore their mechanisms by using kinetically fast electron/hole traps: cyclopropylamine-substituted bases, especially N4-cyclopropylcytosine (CPC), and N2-cyclopropylguanine (CPG). Both biochemical reaction with a variety of photooxidants and electrochemistry show that the modified bases, CPC and CPG, have similar redox properties as the natural DNA bases and are irreversible kinetic traps by ring opening on the picosecond time scale.
\r\n\r\nIn DNA assemblies containing either [Rh(phi)2(bpy')]3+ (Rh) or an anthraquinone derivative (AQ), two high energy photooxidants, appreciable oxidative damage at a distant CPC is observed, which shows that hole migration must involve also the higher energy pyrimidine bases. The damage yield is modulated by lower energy guanine sites on the same or complementary strand. Significantly, the efficiency in trapping at CPC is similar to that at flanking CPG. Thus, HT is not simply a function of the relative energies of the isolated bases, but instead may require orbital mixing among the bases. Hole migration through DNA involves occupation of all the DNA bases with radical delocalization.
\r\n\r\nThe oxidation of CPC via distant photooxidants has been found also to be sensitive to intervening structure and sequences. AQ-modified DNA assemblies of identical base composition but different base sequence have been probed. Single and double base substitutions within A-tracts modulate CPC decomposition. In fact, the entire sequence within the DNA assembly is seen to govern CPC oxidation, not simply the bases intervening between CPC and the tethered photooxidant.
\r\n\r\nThese data are reconciled in a mechanistic model of conformationally gated hole transport through delocalized DNA domains. Oxidation of CPG separated from a tethered photooxidant by A-tracts with a series of lengths over 50 A exhibits a nonmonotonically periodic distance dependence and shows that the domain sizes in the A-tract is 4-5 base pairs. Sequence-dependent DNA structure and dynamics are essential to the transient formation of the domains and hole propagation among the domains. This dynamic, delocalized model provides a basis to reconcile and exploit DNA HT chemistry.
\r\n\r\nJust as long-range hole transport through DNA has now been established, DNA-mediated electron transport has not been as well characterized. Three iridium complexes have therefore been designed in order to initiate both photooxidative and photoreductive reaction of DNA and allow direct comparison between the two. Redox potentials of excited Ir complexes are determined by both triplet energy (E0-0) and ground state redox potentials. Two of the iridium complexes prepared have excited state potentials that are suffcient to oxidize purines, but not pyrimidines. The excited state oxidation potentials of three Ir complexes are around -1.0 V and would be able to reduce DNA pyrimidines. Both CPC and CPG in DNA can be decomposed by photoirradiation with the noncovalently bound iridium complexes. In particular, two of the complexes have the potential to probe oxidation of purines and reduction of pyrimidines in DNA.
\r\n\r\nStudies were also conducted using one of the iridium complexes covalently tethered to DNA oligonucleotides. Hence the metal complex serves as both a photooxidant and photoreductant in the study of DNA-mediated hole and electron transport. In the Ir-tethered DNA assemblies, a metal complex stabilizes the DNA duplex through its intercalative, functionalized dppz ligand. Cyclopropylamine-substituted bases, CPC and CPG, are used as kinetic fast electron and hole traps to probe the resulting charge migration processes after direct photoirradiation of the assemblies. Reductive decomposition of CPC via ET as well as the oxidation of CPG via HT is observed. Thus, the iridium tethered DNA containing cyclopropylamine-substituted bases provides a unique model system to explore the two DNA-mediated charge transport processes through the same DNA bridges. For the first time, ET and HT can be initialized by the same photoredox probe employing the identical electronic interaction mode with DNA.
\r\n\r\nA flash quench technique was also applied to Iridium-tethered DNA in order to generate the ground state photoreductant and initiate photoreduction using 5'-bromo-uridine (BrU) as the electron trap. Efficiencies of BrU reduction in Ir-DNA upon flash quench technique was found to be comparable to that of CPG oxidation upon direct photoirradiation of Ir-DNA. Furthermore, in Ir-tethered DNA assemblies containing CPG or BrU as either the hole or electron trap, the sequence dependence of HT versus ET through an A-tract was examed. When CPG and BrU are placed in either purine or pyrimidine strands in A-tract, decomposition of both modified bases are observed. Thus, transient electron occupancy during ET, as well as hole occupancy during HT, are distributed onto both purine and pymidine strands in A-tract. Additionally, BrU decomposes in a more efficient fashion when it is located on a thyime-containing strands, which indicates that DNA-mediated ET prefers to pyrimidine strands rather than purine strands.
\r\n", "doi": "10.7907/VZ0J-W403", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2205", "collection": "thesis", "collection_id": "2205", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05282007-225441", "primary_object_url": { "basename": "keathesis75.pdf", "content": "final", "filesize": 2877981, "license": "other", "mime_type": "application/pdf", "url": "/2205/1/keathesis75.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Fundamental Mechanisms and Biological Applications of DNA-Mediated Charge Transport", "author": [ { "family_name": "Augustyn", "given_name": "Katherine Emily", "clpid": "Augustyn-Katherine-Emily" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Collier", "given_name": "C. Patrick", "orcid": "0000-0002-8198-793X", "clpid": "Collier-C-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The Pi-stacked array of heterocylic aromatic DNA base pairs provides an intriguing medium for facilitating the transport of migrating charges. The mechanism of hole transport through this dynamic molecule has been extensively investigated using a wide range of techniques. In particular, our group has taken advantage of the octahedral metal complexes of rhodium (III) and ruthenium (II) to probe charge transport reactions through DNA at long range. These intercalating photooxidants, which are extremely well coupled to the DNA ?-stack, can provide us with mechanistic information through a variety of biochemical and spectroscopic techniques. Here we continue to investigate the mechanism of DNA-mediated charge transport on fast time scales using a variety of hole traps and photooxidants and examine this interesting chemistry in a biological context. DNA-mediated charge transport across three different adenine tracts lengths is monitored using a probe interior to the bridge, N6-cyclopropyladenine, CPA. Upon oxidation, the cyclopropylamine-subsituted deoxynucleoside decomposes rapidly, and the efficiency of decomposition can be used as a kinetically fast measurement of hole occupancy. This trap, incorporated serially across the bridge, can be oxidized by a distally bound photooxidant, [Rh(phi)2(bpy\u2019)]3+ (phi = 9,10-phenanthrenequinone diimmine) without significant attenuation in yield over a distance of 5 nm. These results are consistent with complete delocalization across the DNA bridge. Photooxidation of N2-cyclopropylguanine, CPG, within duplex DNA is used to probe DNA charge transport reactions initiated by the covalently bound photooxidants, [Rh(phi)2(bpy\u2019)]3+ and anthraquinone. Duplexes containing the photooxidant separated from the CPG trap by an increasing number of intervening bases are examined in order to probe DNA charge transport reactions with this kinetically fast hole trap as a function of distance and sequence. Charge transport events through sequences containing various length adenine tracts as well as most mixed sequence bridges do not simply decay exponentially nor geometrically as a function of distance. In particular, for variable-length A-tracts, decomposition decreases in a periodic fashion with increasing distance between the photooxidant and the trap; the period is ~4-5 base pairs. Results obtained from charge injection studies using 2-aminopurine as a fluorescent probe have shown a similar periodic distance dependence. These periodicities are not observed in measurements of oxidative DNA damage using double guanine sites as a slow, irreversible hole trap. Thus, CT through DNA must be probed on multiple time scales to provide mechanistic information. These results are consistent with our model for DNA CT through transient delocalized DNA domains defined by sequence-dependent base pair dynamics. While mechanistic investigations are critical for a fundamental understanding of how charges migrate through DNA, it is important to consider the biological consequences of this process. A biological role for DNA-mediated CT has been investigated in the context of the transcription factor, p53, a tumor suppressor protein involved in myriad cellular pathways such as apoptosis and growth arrest. DNA assemblies containing an anthraquinone photooxidant tethered to the 5\u2019 end of sequences containing p53 binding sites were constructed to examine the binding affinity as a function of photooxidation. We demonstrate that through photoinduced DNA-mediated CT, the p53 protein becomes oxidized and exhibits differential binding for various promoter sequence including Gadd45, p21, and Mdm2. Additionally, insertion of a mismatch intervening between the photooxidant and the p53 binding site serves to attenuate this change in binding affinity associated with photooxidation. MALDI-TOF mass spectrometric analysis of p53 tryptic digests following irradiation of the DNA bound protein provides further evidence that a chemical change occurs, consistent with oxidation of a cysteine residue in the DNA binding domain. Dipyridophenazine complexes of ruthenium (II) have been used extensively to spectroscopically investigate DNA-mediated charge transport. A novel tris heteroleptic dipyridophenazine complex of ruthenium (II), [{Ru(phen)(dppz)(bpy\u2019-his)}{Ru(NH3)5}]5+, containing a covalently tethered ruthenium pentaammine quencher coordinated through a bridging histadine has been synthesized and characterized spectroscopically and biochemically in a DNA environment and in organic solvent. Capable of undergoing intramolecular photoinduced electron transfer, the steady-state and time-resolved luminescence measurements indicate that the tethered-quencher complex is quenched relative to the parent complexes [Ru(phen)(dppz)(bpy\u2019]2+ and [Ru(phen)(dppz)(bpy\u2019-his)]2+ in DNA and acetonitrile. Intercalated into guanine containing DNA, [{Ru(phen)(dppz)(bpy\u2019-his)}{Ru(NH3)5}]5+, upon excitation and intramolecular quenching, is capable of injecting charge into the duplex as evidenced by EPR detection of guanine radicals. DNA-mediated charge transport is also evidenced using a kinetically fast cyclopropylamine-substituted base as a hole trap that undergoes irreversible oxidative ring opening on the picosecond time scale. Guanine oxidation is not observed in measurements using guanine radical as a slow, irreversible hole trap indicating that back electron transfer reactions are competitive with hole injection into the duplex. Moreover, transient absorption measurements reveal a novel photophysical reaction pathway for [{Ru(phen)(dppz)(bpy\u2019-his)}{Ru(NH3)5}]5+ in the presence of DNA, competitive with the intermolecular flash-quench process. These results illustrate the remarkable redox chemistry occurring within a bimolecular ruthenium complex intercalated in duplex DNA.", "doi": "10.7907/AYHS-7Q13", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:3300", "collection": "thesis", "collection_id": "3300", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08312006-131856", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 6311994, "license": "other", "mime_type": "application/pdf", "url": "/3300/1/Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Electrochemical Studies of Electron Transfer in DNA Films with Covalently Tethered Intercalators", "author": [ { "family_name": "Liu", "given_name": "Tao", "clpid": "Liu-Tao" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Collier", "given_name": "C. Patrick", "clpid": "Collier-C-P" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The base stack within double-helical DNA provides an efficient pathway for charge transport. This DNA-mediated charge transport chemistry has been shown to be exquisitely sensitive to minor perturbations in DNA structure and base stacking both in solution and on surfaces. As a result, electrochemical studies on DNA-modified electrodes may provide a novel approach to the development of sensitive, but inexpensive DNA sensing devices. Using intercalated, covalently bound daunomycin as a redox probe, we have examined ground state charge transport in DNA films on gold electrodes. DNA-mediated electron transfer is found to occur over a distance as long as 100 \u00c5 in the film. Moreover, while the introduction of one or even two breaks in the sugar-phosphate backbone yields no detectable effect on electron transfer, a CA base-pair mismatch significantly attenuates the electron transfer yield. These results confirm that the base pair stack is the pathway for DNA mediated charge transfer, not the sugar-phosphate backbone. Based on these studies, we have developed a method to electrochemically monitor the trapping of double- stranded DNA with a 6-base overhang on a gold electrode modified with double- stranded DNA probes containing a complementary overhang. The trapping of the double-stranded target can be monitored by the reduction of daunomycin crosslinked to the target. A CA mismatch in the target duplex can also be detected by the diminished reduction signal.
\r\n\r\nIt has been shown that the electronic coupling between an intercalator and the pi-stack of DNA is critically important for the reduction of the intercalator through DNA-mediated charge transport. Using covalently tethered anthraquinone derivatives as the redox probe, we have investigated also the influence of the binding mode of the intercalator on its reduction in DNA films. The results of these studies underscore the importance of direct interaction between the redox probe and the pi-stack in order to observe efficient DNA-mediated electrochemistry through DNA films. These studies have also shown that the covalent linkage has a significant effect on the intercalation of the probe to the base stack of DNA.
\r\n\r\nIn an effort to develop a redox probe that has effective electronic coupling with the pi-stack while being covalently tethered to a DNA strand with a stable linkage, we have crosslinked Nile blue, a redox active DNA intercalator with high DNA binding affinities, to the 5\u2019-end of oligonucleotides. The covalently tethered Nile blue is shown to be sensitive probe for DNA-mediated electron transfer on the gold surface. An intervening CA mismatch has been detected in both tightly packed and loosely packed films of DNA\u2013Nile blue conjugate. We have also coupled the reduction of Nile blue to an electrocatalytic cycle involving freely diffusing ferricyanide, which significantly enhances the sensitivity to intervening mismatches. Furthermore, using Nile blue as the covalently tethered probe, we have developed a method for DNA mismatch detection that eliminates sample modification and has a potential for high throughput assays. These studies may provide a practical approach to diagnostic devices for mutation detection with high sensitivity and low expense.
", "doi": "10.7907/MMSW-4A41", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:2655", "collection": "thesis", "collection_id": "2655", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06202006-113417", "primary_object_url": { "basename": "Ceres_PhD_Thesis.pdf", "content": "final", "filesize": 12510610, "license": "other", "mime_type": "application/pdf", "url": "/2655/1/Ceres_PhD_Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Electron Transfer at DNA-Modified Electrodes", "author": [ { "family_name": "Ceres", "given_name": "Donato Marino", "clpid": "Ceres-Donato-Marino" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The DNA pi stack provides an efficient pathway for transport of electron and electron holes. Ground-state electron transport is furthermore extremely sensitive to subtle DNA structural perturbations, such as a single base mismatch, that alter pi-stacking. As a result, DNA-modified electrodes have allowed the development of highly sensitive diagnostic devices for the detection of base mismatches, lesions, and mutations. We have been able to apply DNA-mediated charge transduction, using a methylene blue/ferricyanide electrocatalytic cycle, in a DNA chip format for the detection of a single base mismatches at a microelectrode. Electrocatalysis is detected at DNA-modified electrodes down to 40 um electrode in diameter, where 108 DNA molecules are responsible for the electron transduction. This exquisite sensitivity both for mismatch detection irrespective of sequence context and to a small number of molecules is an important requisite for the development of a device able to detect multiple genetic variations in the absence of DNA amplification.
\r\n\r\nWe have also investigated in detail the electrochemical properties of DNA films. DNA is a highly charged molecule and, when self-assembled on a gold surface in a dense array, its properties are similar to those of polyelectrolyte films. We have found that the structure of the DNA film is sensitive to ion concentration and identity. Variations of the electrostatic potential across the film can sensitively affect both thermodynamics and kinetics of redox reporters incorporated in the film. Methylene blue reduction in the DNA film occurs via a two electron, one proton process. The Pourbaix diagram is linear in the case of a monovalent anionic buffer, while it is curved in phosphate buffer. Electron transfer kinetics are also affected by the relative concentration of divalent anions: at low pH the film is compressed in the linker portion and the rate of electron transfer is faster. Based on this understanding of the electrostatic balance inside the DNA film, a new analytical tool for monitoring hybridization events on gold surfaces has been developed using electrochemical impedance spectroscopy of ferricyanide.
\r\n\r\nIn order to explore the electron transport properties of DNA films mechanistically scanning tunneling microscopy (STM) has also been employed. These experiments provide a first opportunity to examine DNA conductivity under physiological conditions. These STM experiments on DNA films show that DNA, when perpendicularly oriented with respect to the surface, is coupled to the STM tip and the local density of states contribute to the measured tunneling current. At positive biases, when the surface is positive, the DNA is tilted towards the surface and as a result decoupled from the tip; the DNA appears \"transparent\" and the underlying surface instead is imaged. Also important is the integrity of the base stack. When the percentage of DNA duplexes containing a single base mismatch in the film is increased, the conductivity of the film decreases. The STM tip, being held at a constant current, approaches the DNA film until, at a critical mismatch content, the tip must penetrate the film and image resolution is lost. The current versus voltage characteristic of the DNA film has furthermore been determined through a new scanning tunneling spectroscopic technique that provides highly stable and reproducible measurements. We find that DNA duplex films under physiological conditions exhibit negative differential resistance, which is a feature that is typical of resonant electron tunneling via energetically localized molecular orbitals. This observation provides an experimental evidence for the existence of localized states within the DNA HOMO-LUMO gap that can be responsible for the ground state electron transport observed in electrochemical experiments.
", "doi": "10.7907/vdyv-zf71", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2528", "collection": "thesis", "collection_id": "2528", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06092006-062410", "primary_object_url": { "basename": "thesis3.pdf", "content": "final", "filesize": 7605036, "license": "other", "mime_type": "application/pdf", "url": "/2528/1/thesis3.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Synthesis and Applications of Bulky Rhodium(III) Intercalators for the Recognition of DNA Mismatches", "author": [ { "family_name": "Hart", "given_name": "Jonathan Ross", "orcid": "0000-0002-3905-225X", "clpid": "Hart-Jonathan-Ross" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "orcid": "0000-0002-6610-4414", "clpid": "Peters-J-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The recognition of DNA base mismatches is of considerable interest for both the diagnosis and treatment of mismatch repair-deficient cancers. Two new mismatch recognition complexes have been synthesized. The first, [Rh(bpy)2(phzi)]3+ (phzi=benzo[a]phenazine-5,6-quinone diimine), recognizes DNA mismatches with high specificity and affinity, 1 x 107 Mm-1, two orders of magnitude stronger than [Rh(bpy)2(chrysi)]3+ (chrysi=chrysene-5,6-quinone diimine), the parent complex that binds single thermodynamically-destabilized base-mismatch sites in duplex DNA. The second, [Rh(bqdi)2(chrysi)]3+, is able to recognize more stable mismatches such as the G-G mismatch.
\r\n\r\nThese complexes have been applied in a variety of ways. A method has been developed for the discovery of new single nucleotide polymorphisms, SNPs, within a sequence of interest amplified from pooled genomic DNA. SNPs are readily detected using these mismatch selective molecules without false positives; allele frequencies as low as 0.05 can be detected.
\r\n\r\nUpon photoexcitation, the rhodium(III) diimine complexes cleave DNA by hydrogen atom abstraction from the sugar to yield 3'-phosphate terminated DNA that is inactive for enzymatic modification. This 3'-phosphate can be removed using T4-polynucleotide kinase opening up the possibility of enzymatic modification at the site of rhodium cleavage. The cleavage site can be fluorescently labeled. Terminal transferase can also be used to attach a homopolymer tail tagging the damage site, allowing the amplification of the DNA up to the damaged site.
\r\n\r\nThis assay can also be employed towards the development of early cancer diagnostics. Some cancers are deficient in the repair of DNA base mismatches. As a consequence, these cells have an increased number of mismatches within their genome. These mismatches in extracted genomic DNA were cleaved using mismatch-specific rhodium complexes. The cleavage sites were labeled with radioactivity, allowing the number of mismatch sites to be quantitated. A significant number of sites were cleaved in the mismatch repair deficient DU145 cell line, 1 base/3000 bp, while no sites were cleaved in the mismatch repair proficient cell line SW620. This method may present a new method for the detection of mismatch repair deficiency.
\r\n\r\nThese mismatch-specific complexes also are shown to have an antiproliferative effect on mismatch repair deficient cell lines. Mismatch repair deficiency is a contributing factor in both hereditary and sporadic human cancers. Both [Rh(bpy)2(chrysi)]Cl3 and [Rh(bpy)2(phzi)]Cl3 show a stronger antiproliferative effect against MMR deficient cells than proficient cells. Effects of stereoisomers, incubation time, and UV irradiation are also demonstrated.
", "doi": "10.7907/sh3b-2f25", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:1856", "collection": "thesis", "collection_id": "1856", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05182004-181406", "primary_object_url": { "basename": "TitlePage.pdf", "content": "final", "filesize": 48514, "license": "other", "mime_type": "application/pdf", "url": "/1856/2/TitlePage.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Oxidative DNA Damage by Long-Range Charge Transport", "author": [ { "family_name": "Delaney", "given_name": "Sarah", "orcid": "0000-0002-8366-3808", "clpid": "Delaney-Sarah" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ever since the elucidation of the double helical structure of DNA, it has been proposed that the stack of base pairs within the double helix may mediate charge transport (CT) reactions. In fact, CT through DNA can result in chemistry at a distance, yielding oxidative DNA damage at a site remote from the bound oxidant. DNA CT chemistry depends upon coupling within the stacked base pair array, and this chemistry is remarkably sensitive to sequence-dependent DNA structure and dynamics. Using a variety of octahedral transition metal complexes, DNA CT has been probed to explore mechanistic considerations and biological possibilities.
\r\n\r\nInteractions with DNA by a family of ruthenium(II) complexes bearing the dipyridophenazine (dppz) ligand or its derivatives have been examined. An intercalative binding mode has been established based on luminescence enhancements in the presence of DNA, excited state quenching, fluorescence polarization values and enantioselectivity. Oxidative damage to DNA by these complexes using the flash/quench method has also been examined. A direct correlation between the amount of guanine oxidation obtained via DNA CT and the strength of intercalative binding was observed. These results support the importance of close association and intercalation for DNA-mediated CT. Electronic access to the DNA base pairs, provided by intercalation of the oxidant, is a prerequisite for efficient CT through the DNA pi-stack.
\r\n\r\nUsing polypyridyl ruthenium complexes, a reductive flash/quench scheme in DNA has also been explored. The flash/quench scheme previously utilized in DNA studies involves an oxidative quencher and allows for examination of electronic hole transport through DNA. In contrast, a reductive flash/quench technique would allow for direct observation of electron transport through the base stack. In our studies, p-methoxydimethylaniline and potassium iodide have proven to be effective reductive quenchers of dipyridophenazine complexes of ruthenium. However, by transient absorption spectroscopy, high performance liquid chromatography, gel electrophoresis, and electron paramagnetic resonance we are unable to observe any DNA reduction products with the ruthenium complexes examined. Rates of back electron transfer may in fact be faster than trapping of the anion radical, thus hindering observation of long-range damage.
\r\n\r\nThe oxidative flash/quench technique was applied in probing DNA CT in a range of DNA assemblies containing a tethered ruthenium intercalator and methylindole (M), a low potential nucleobase analog, where radical formation at a distance as a function of DNA sequence could be examined both by laser spectroscopy and biochemical methods. Hole injection and subsequent formation of the methylindole radical cation were observed at a distance of over 30 \u0160at rates > 10e7 s-1 in assemblies containing no guanine bases intervening the ruthenium intercalator and GMG oxidation site. Radical yield was, however, strikingly sensitive to an intervening base mismatch; no significant methylindole radical formation was evident with an intervening AA mismatch. Also critical is the sequence at the injection site; this sequence determines initial hole localization and hence the probability of hole propagation. With guanine rather than inosine near the site of hole injection, decreased yields of radicals and long-range oxidative damage are observed. The presence of the low energy guanine site in this case serves to localize the hole and increase the probability of back reaction at the injection site therefore diminishing CT through the base pair stack.
\r\n\r\nDNA assemblies containing a pendant dppz complex of Ru(II) along with two oxidative traps, a site containing the nucleoside analog methylindole (5?-GMG-3?) and a 5?-GGG-3? site, were constructed to explore charge equilibration across the base pair stack. In these assemblies the base radicals form with a rate of 10e7 s-1. Interestingly, the rate of base radical formation does not change upon the addition of a second radical trap, the 5?-GGG-3? site; however the yield of methylindole oxidation is significantly lower. This observation indicates that the 5?-GGG-3? site is effective in competing for the migrating charge and provides a second trapping site. Importantly, switching the orientation of the two trapping sites does not affect the yield of oxidized products at either site. Therefore, in DNA both forward and reverse charge transport occur so as to provide equilibration across the duplex on a time scale that is fast compared to trapping at a particular site. Further evidence of charge equilibration results from incorporating an intervening base-stacking perturbation and monitoring the fate of the injected charge. These experiments underscore the dynamic nature of DNA charge transport and reveal the importance of considering radical propagation in both directions along the DNA duplex.
\r\n\r\nDNA conjugates containing adjacent duplex and guanine quadruplex assemblies have been designed to explore CT into quadruplex architectures. The quadruplex assemblies have been characterized structurally using circular dichroism and by assaying for chemical protection. Using an intercalating rhodium photooxidant, noncovalently bound or tethered to the duplex end, oxidizing radicals are found to be trapped in the folded quadruplex. Damage is observed almost exclusively at the external tetrads of the quadruplex. Little damage of the center tetrad is observed, due most likely to lowered efficiency of radical trapping within the quadruplex core. This pattern of damage is distinct from that observed for repetitive G sequences within duplex DNA. The data indicate, furthermore, that in the conjugates examined, the guanine quadruplex provides a more effective trap than a 5?-GG-3? guanine doublet within duplex DNA. Additionally, within these assemblies, sufficient base-base overlap must exist at the duplex/quadruplex junction to allow for charge migration. This funneling of damage to the quadruplex, as well as the unique pattern of damage within the quadruplex, requires consideration with respect to the analysis of oxidative DNA damage within the cell.
", "doi": "10.7907/9Q0X-TZ17", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1744", "collection": "thesis", "collection_id": "1744", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05122004-052751", "primary_object_url": { "basename": "title.pdf", "content": "final", "filesize": 44549, "license": "other", "mime_type": "application/pdf", "url": "/1744/7/title.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Structure and Reactivity of Metal Complexes Bound to DNA", "author": [ { "family_name": "Bhattacharya", "given_name": "Pratip K.", "orcid": "0000-0002-0625-252X", "clpid": "Bhattacharya-Pratip-K" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" }, { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Establishing correlations among structure, dynamics and reactivity is a fundamental problem in biological chemistry. Here, this problem is explored in the context of the design and reactivity of different metallointercalators bound to DNA.
\r\n\r\nFirst, the effects of intervening mismatches on DNA structure, dynamics and DNA charge transport reactivity is examined. The \u03c0-stacked DNA base pairs mediate charge transport chemistry over long molecular distances in a reaction that is exquisitely sensitive to DNA sequence dependent conformation and dynamics. To examine the long-range charge transport as a function of intervening base mismatches, a series of DNA oligonucleotides were synthesized that incorporate a ruthenium intercalator, [Ru(phen)(bpy')(dppz)]\u00b2\u207a (phen = 1,10 phenanthroline; bpy' = 4-butyric acid-4'-methylbipyridine; dppz = dipyrido[3,2-a:2',3'-c]phenazine) linked covalently to the 5' terminus of one strand and containing two 5'-GG-3' sites in the complementary strand. Single base mismatches were introduced between the two guanine doublet steps, and the efficiency of transport through the mismatches was determined through measurements of the ratio of oxidative damage at the guanine doublets distal versus proximal to the intercalated ruthenium oxidant. Differing relative extents of guanine oxidation were observed for the different mismatches. The damage ratio of oxidation at the distal versus proximal site for the duplexes containing different mismatches varies in the order GC ~ GG ~ GT ~ GA > AA > CC ~ TT ~ CA ~ CT. The extent of distal/proximal guanine oxidation in different mismatch-containing duplexes was then compared with the helical stability of the duplexes, electrochemical data for intercalator reduction on different mismatch-containing DNA films, and base-pair lifetimes for oligomers containing the different mismatches derived from \u00b9H NMR measurements of the imino proton exchange rates. The exchange kinetics of the imino protons were measured from selective longitudinal relaxation times, and the effect of the mismatch was observed on the base pair lifetime up to a distance of two neighboring base pairs. The overall order of base-pair lifetimes in the selected sequence context of the base pair was as follows: GC > GG > AA > CC > TT. While a clear correlation is evident both with helix stability and electrochemical data monitoring reduction of an intercalator through DNA films, guanine damage ratios was found to correlate most closely with base-pair lifetimes. These results underscore the importance of base dynamics in modulating long-range charge transport through the DNA base-pair stack.
\r\n\r\nIn a related \u00b9H NMR structural study of the ruthenium intercalator, [Ru(phen)(bpy')(dppz)]\u00b2\u207a covalently tethered to a short eightmer DNA duplex, d(ACGAGCAC)\u2022d(GTICTCGT) with a nine carbon linker, the type of construct used in charge transport experiments, a very fast exchange was observed. Comparison of the NOESY data obtained from the NMR study of this system and control samples comprising of the duplex with only linker and the duplex alone, led to the conclusion that the nine carbon linker is positioned between the second and fourth bases from the point of its origin. The absence of any site specificity of the metal complex in the oligonucleotide complicates the structural characterization by NMR study. This led us to conceive of a more general strategy of obtaining structural information of metal complexes that bind non-specifically to DNA based on paramagnetic NMR.
\r\n\r\nThe selective paramagnetic relaxation of oligonucleotide proton resonances of two short self-complementary oligonucleotides; d(GTCGAC)\u2082 and d(GTGCAC)\u2082 by Ni(phen)\u2082(L)\u00b2\u207a where L= dipyridophenazine (dppz), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) and phenanthrenequinone (phi) was examined to obtain structural insight into the non-covalent binding of these metal complexes to DNA. In the oligonucleotide d(GTCGAC)\u2082, preferential broadening of the G1H8, G4H8, T2H6, and C3H6 proton resonances was observed with Ni(phen)\u2082(dppz)\u00b2\u207a, Ni(phen)\u2082(dpq)\u00b2\u207a and Ni(phen)\u2082(phi)\u00b2\u207a. In the case of the sequence d(GTGCAC)\u2082, where the central two bases are juxtaposed from the previous one, preferential broadening was observed instead for the A5H2 proton resonance. Thus, a subtle change in the sequence of the oligonucleotide can cause significant change in the binding location of the metal complex in the oligonucleotide. Owing to comparable changes for all metal complexes and sequences in broadening of the thymine methyl proton resonances, the switch in preferential broadening was attributed to a change in site location within the oligomer rather than to an alteration of groove location. Therefore, even for DNA-binding complexes of low sequence-specificity, distinct variations in binding as a function of sequence are apparent and can be monitored using paramagnetic probes.
\r\n\r\nFinally, \u00b9H NMR spectroscopy was employed to study the binding of [Rh(bpy)\u2082chrysi]\u00b3\u207a (chrysi = 5,6-chrysenquinone diimine), a metal complex which specifically targets mismatches, to a ninemer oligonucleotide d(GCCTCAGGC)\u2082 containing centrally placed CC mismatch. Evidence supports intercalation by the metal complex within the mismatch site (i) upfield chemical shifts and significant broadening of the chrysi resonances and (ii) an increase in duplex melting temperature in the presence of the metal complex. To simplify the NMR spectra, the \u0394 isomer of [Rh(d\u2088\u208bbpy)\u2082chrysi]\u00b3\u207a was employed in NMR experiments with DNA. A break in the connectivity in the NOE walk is observed between T\u2084 and C\u2085, thereby marking the binding site of the metal complex at the CC mismatch. Intermolecular NOE's place the metal complex in the major groove of the oligonucleotide.
\r\n\r\nThus through a series of experiments in this thesis, attempts have been made to correlate the structure and dynamics of metal complexes bound to DNA. Truly, metal complexes bound to DNA provide an interesting system to study structure, function and dynamics in a single package.
", "doi": "10.7907/ZANY-J082", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1305", "collection": "thesis", "collection_id": "1305", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04082004-161537", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 5218662, "license": "other", "mime_type": "application/pdf", "url": "/1305/1/thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Fundamental Aspects of DNA-Mediated Charge Transport", "author": [ { "family_name": "Williams", "given_name": "Tashica Tr\u00e9shun", "clpid": "Williams-Tashica-Tr\u00e9shun" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The \u03c0-stacked array of DNA base pairs has fascinated scientists since its structural delineation. Here are described fundamental studies to probe how this \u03c0-stacked array mediates DNA charge transport (CT). Intercalators, such as dipyridophenazine (dppz) complexes of ruthenium and phenanthrenequinone diimine (phi) complexes of rhodium, serve as powerful and systematic probes of DNA CT in these studies.
\r\n\r\nIn a series of rhodium-tethered DNA assemblies, with varying A/T sequences intervening between guanine doublet sites (5'-GG-3'), long-range oxidative DNA damage is examined. The guanine doublet sites are known as sites of low oxidation potential in DNA, and the rhodium complex serves as a spatially separated, potent photooxidant. Although these studies are inconsistent with a mechanism involving guanine hopping and tunneling through A/T sequences, these data illustrate that the sequence of bases is an important determinant in attenuating oxidative damage yields of CT. Based on these data, we propose hopping among domains defined by sequence-dependent structure. Additional studies are also described using rhodium-tethered DNA assemblies to examine how different ionic distributions around the DNA duplex modulate DNA CT. In the rhodium-DNA conjugates, differences in long-range oxidative damage yield were observed depending on the position of pendent charges on the oligomer.
\r\n\r\nA direct comparison of DNA CT utilizing a variety of oxidants has also been performed. CT is assayed both through determination of the yield of oxidative guanine damage and, in derivative DNA assemblies, by analysis of the yield of a faster oxidative trapping reaction, ring-opening of N2-cyclopropylguanine (CPG) within the DNA duplex. We find clear differences in oxidative damage ratios at the distal versus proximal 5'-GG-3' depending upon the photooxidant employed. There is also a correlation seen between absolute yield of oxidative damage and distal/proximal damage ratio; photooxidants that produce higher distal/proximal damage ratios have lower yields. These differences observed among photooxidants as well as the complex distance dependence are attributed to differences in rates of back electron transfer (BET).
\r\n\r\nA study of the overall effect of bridge energetics on DNA CT has also been performed by constructing rhodium-DNA assemblies containing varying numbers of inosine, a guanine base analog with a higher oxidation potential, between two 5'-GG-3' sites. For the rhodium conjugates, only a slight diminution in distal oxidative yield with increasing distance is observed, suggesting direct charge injection by rhodium into higher energy sites of the intervening bridge.
\r\n\r\nThese results, taken together, provide insight into salient parameters that govern DNA CT, in particular how energetics, charge distribution, and sequence-dependent DNA structure and dynamics modulate charge migration through DNA.
", "doi": "10.7907/83ec-2t89", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2038", "collection": "thesis", "collection_id": "2038", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05252003-231704", "primary_object_url": { "basename": "00Title_Page.pdf", "content": "final", "filesize": 127141, "license": "other", "mime_type": "application/pdf", "url": "/2038/1/00Title_Page.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Spectroscopic Characterization of DNA-Mediated Charge Transfer", "author": [ { "family_name": "Treadway", "given_name": "Christopher Ryan", "clpid": "Treadway-Christopher-Ryan" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The DNA double helix provides a well-characterized molecular pi stack in which charge transfer rates and efficiencies may be examined. We investigated the phenomenon in a series of DNA duplexes modified with various photo- and redox-active species.
\r\n\r\nCharge transfer dynamics through DNA were distance independent for a series of duplexes modified with 7-deazaguanine and a covalently attached ethidium chromophore. The decay times were grouped into two components: (1) injection of charge into the DNA base stack and (2) a correlated charge transfer corresponding to the reorientation of ethidium within the duplex.
\r\n\r\nUsing the modified bases 2-aminopurine and 7-deazaguanine, intrastrand charge transfer through DNA was observed on the picosecond timescale. Charge transfer rates and quenching yields were also dependent on the reaction?s driving force and the composition of the intervening base stack.
\r\n\r\nThe efficiency of photooxidation of 7-deazaguanine by a ruthenium(II) intercalator in DNA over 7?14 angstroms was shallow and dependent on the chirality of the covalently attached metallointercalator. Nanosecond to subnanosecond decay rates were measured, and the spectroscopic signature of a charge transfer intermediate was observed.
\r\n\r\nA series of ruthenium(II) intercalators with high oxidation potentials was created. Redox reactivity of the compounds with DNA did not correlate directly with oxidation potential and was dependent on DNA binding and luminescence quenching abilities. Thus, redox potential may not be used as the sole predictor of reactivity with the base stack of DNA.
\r\n\r\nFinally, the binding of ruthenium(II) and rhodium(III) intercalators to DNA was investigated with CD and NMR spectroscopies. Data confirmed that metallointercalators do not preferentially bind next to each other along the double helix. Hence, direct contact of reactants is not responsible for fast and efficient charge transfer between metallointercalators bound noncovalently to DNA.
\r\n\r\nThese studies have provided direct measurements of the dynamics of DNA-mediated charge transfer and proven, once again, that the DNA pi stack facilitates fast and efficient charge transfer. Most importantly, the stacking and dynamics of the reactants and DNA bases were found to affect the charge transfer behavior.
", "doi": "10.7907/KMWD-4M75", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:3194", "collection": "thesis", "collection_id": "3194", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08222002-162219", "primary_object_url": { "basename": "ack,abs,TOC.pdf", "content": "final", "filesize": 162916, "license": "other", "mime_type": "application/pdf", "url": "/3194/1/ack,abs,TOC.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Electrochemical Sensors Based on DNA-Mediated Charge Transport Chemistry", "author": [ { "family_name": "Boon", "given_name": "Elizabeth Marshall", "orcid": "0000-0003-1891-839X", "clpid": "Boon-Elizabeth-Marshall" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Anson", "given_name": "Fred C.", "clpid": "Anson-F-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The base pair stack within double helical DNA provides an effective medium for charge transport. The \u03c0-stacked DNA base pairs mediate charge transport chemistry over long molecular distances in a reaction that is exquisitely sensitive to DNA sequence-dependent conformation and dynamics. This sensitivity to minor perturbations in DNA structure and base stacking makes DNA-mediated charge transport chemistry an ideal platform for DNA sensing. Electrochemical methods through DNA-modified electrode surfaces that exploit this sensitivity for efficient biosensing are described. Gold electrodes are modified with DNA double helices and used to monitor the electrochemistry of bound redox-active intercalators. The efficiency of electrochemical reduction of the intercalated redox-probe, in a DNA-mediated reaction, provides an indicator of base stacking within the surface-bound duplexes. Perfectly stacked DNA is capable of mediating the electrochemical reduction, while duplexes containing \u03c0-stacking perturbations, such as single base mismatches, do not support current flow to the intercalator.
\r\n\r\nAll single base mismatches, including thermodynamically stable GT and GA mismatches, as well as many common base damage products can be detected within DNA and DNA/RNA hybrid duplexes using this assay. Moreover, mismatches can be detected as a small percentage of a perfectly matched film, making it possible to detect mutations associated with genetic disorders in only a small fraction of cells. This assay is also compatible with DNA based chip technology. Furthermore, electrochemistry at DNA films is found to provide a novel and sensitive method for probing protein dependent changes in DNA structure and enzymatic reactions.
\r\n\r\nThe efficient transport of charge through self-assembled monolayers of thiol-terminated duplexes on gold therefore offers an extremely sensitive probe for the integrity of DNA sequences. Completely new approaches to single base mismatch detection as well as assaying protein-DNA interactions and reactions on surfaces are now available. This technology is generally applicable as a tool for directly measuring base pair stacking in nucleic acid duplexes.
", "doi": "10.7907/PFXM-7M76", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:6782", "collection": "thesis", "collection_id": "6782", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01262012-090632523", "primary_object_url": { "basename": "Copeland_kd_2002.pdf", "content": "final", "filesize": 75650200, "license": "other", "mime_type": "application/pdf", "url": "/6782/1/Copeland_kd_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "The Reactions of Metallointercalator-Peptide Conjugates and DNA", "author": [ { "family_name": "Copeland", "given_name": "Kimberly Davis", "clpid": "Copeland-Kimberly-Davis" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A family of metallointercalator-peptide conjugates for reaction with DNA has been constructed. In these chimeras, the metallointercalator provides binding affinity for\r\nDNA and the peptide contributes reactivity. With the goal of creating an artificial nuclease, we have tethered metal-binding peptides to a sequence neutral intercalator,\r\n[Rh(phi)_2bpy']^(3+) (phi = phenanthrenequinone diimine, bpy' = 4-butyric acid-4' -methyl-2,2'-bipyridine). This is a general strategy, and we have observed Zn^(2+)-promoted\r\ncleavage of plasmid DNA with widely different peptides: a designed helical peptide with histidine residues, and a hairpin peptide modeled after the active site of the BamHI\r\nendonuclease. To optimize the peptide composition of our artificial nuclease we created a library of 16,000 conjugates, but no new active conjugates were identified with this combinatorial strategy. To achieve oxidative cleavage of the DNA backbone we have also used our intercalator-peptide conjugates to deliver copper to DNA. Tethered peptides containing histidine residues promote oxidative strand scission in DNA restriction fragments and oligonucleotides in the presence of Cu^(2+) and a reducing agent. Importantly, by comparing the photocleavage pattern of the rhodium intercalator with the copper cleavage pattern of the metal-binding peptide, the interactions of the conjugate with DNA could be dissected. Finally, short peptides were tethered to [Ru(phen)(bpy')(dppz)]^(2+) \r\n(phen = 1,10-phenanthroline, dppz = dipyridophenazine) to\r\ncreate fluorescent DNA crosslinking agents. Through a flash-quench reaction, the ruthenium intercalator generates guanine radicals in a DNA duplex. These guanine\r\nradicals can react with water or oxygen, but also with tethered peptides to produce pennanent DNA-peptide crosslinks. The DNA-peptide crosslinks were detected by gel\r\nelectrophoresis and absorbance measurements, and characterized by mass spectrometry. Although they have low affinity for DNA, untethered peptides could also be crosslinked to DNA using the ruthenium chemistry. The peptide composition influences conjugate binding and the extent and pattern of crosslinking; indeed, positively charged residues were essential for effective crosslinking. Although the flexibility of our tethered peptides\r\nis an obstacle to the rational design of reactive conjugates, we have demonstrated that peptides can mediate a variety of reactions if delivered to DNA by metallointercalators.", "doi": "10.7907/MR9T-7W57", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6797", "collection": "thesis", "collection_id": "6797", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312012-114925650", "primary_object_url": { "basename": "Nunez_me_2002.pdf", "content": "final", "filesize": 51836776, "license": "other", "mime_type": "application/pdf", "url": "/6797/1/Nunez_me_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Oxidation of DNA by Long-Range Charge Transport", "author": [ { "family_name": "Nu\u00f1ez", "given_name": "Megan Elizabeth", "clpid": "Nu\u00f1ez-Megan-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ever since the double helical structure of DNA was elucidated, it has been proposed that charge might move through the stacked base pairs of the double helix because of the electronic coupling of the \u03c0 orbitals of the nucleotide bases with neighboring bases. Here it is demonstrated that electronic \"holes\" generated\r\nby a one-electron oxidation of DNA can result in permanent lesions on guanine bases up to 200 \u00c5 away from the intercalating oxidant as a result of such charge\r\nmigration. Both rhodium and ruthenium complexes, covalently tethered to the 5' end of a double-stranded oligonucleotide and intercalated into the base stack, can with photoactivation promote oxidation of guanines in 5'-GG-3' sites over this distance. Since charges can move efficiently through the DNA oligonucleotides, it was important to characterize this reaction in more detail, and to extend observations of charge transport through DNA to larger and more complicated DNA assemblies that more closely mimic its structure in vivo.
\r\n\r\nLong-range oxidative damage to guanine doublets in DNA is shown to compete for oxidation with other reactions, such as the repair of thymine dimers. When both thymine dimer lesions and guanine doublets are present, both can be\r\noxidized by a photoexcited rhodium complex, although each in lower yield than in the absence of the other. While the 5-GG-3' may represent the thermodynamically favored site for oxidative reaction, repair of the thymine dimer appears to be kinetically more favorable. Therefore electronic \"holes\" generated on genomic DNA might not of necessity cause DNA damage, but could also be funneled onto proteins or other oxidizible sites.
\r\n\r\nUsing a variety of intercalating photooxidants targeted to a specific site on a restriction fragment by an appended triplex-forming oligonucleotide, the upper distance limits and sequence effects on long-range charge transfer through DNA were examined. Charge migration occurs in both directions from the intercalator and on both DNA strands of the target, but the oxidation is significantly more\r\nefficient to the 3' side of the triplex, over 25-38 base pairs. When intercalators were tethered directly to the 5' terminus of the triplex-forming strand as opposed\r\nto the center, significant amounts of oxidative damage was generated only in the immediate vicinity of the intercalation site, suggesting that the base stack is\r\ndistorted at the 5' end of the triplex region in the duplex/triplex junction. Targeting of photooxidative damage by triplex formation extends previous studies of long-range charge transport to significantly longer DNA sequences through a strategy that does not require covalent attachment of the photooxidant to the DNA being probed.
\r\n\r\nWithin eukaryotic cells most DNA is packaged as nucleosome core particles, made up of ~146 base pairs of DNA wrapped around a core of histone proteins. Photoexcited rhodium complexes were also used to explore charge\r\ntransport through DNA within these structures. Although histone proteins inhibit intercalation of a noncovalent rhodium complex, they do not prevent oxidation of\r\n5'-GG-3' sites, the signature of oxidative charge transport through DNA. Furthermore, some of these sites are not directly accessible to a solution-bound oxidant due to his tones in the major groove, and thus they must be oxidized from a distance. Therefore, although the structure of the nucleosome core particle generally protects DNA from damage from solution-borne molecules, it does not protect the DNA from charge transfer damage through the base pair stack. In\r\nsupport of this assertion, guanine bases within nucleosomal DNA were oxidized at a distance of over 23 base pairs from a covalently-tethered rhodium intercalator.
\r\n\r\nThe environment within the cell nucleus contains a variety of other proteins and small molecules that could potentially influence the migration of charge through DNA. Using the rhodium photochemistry, the oxidation of\r\nguanine by photoexcited rhodium complexes inside of nuclei from cultured human cells was examined and compared with the oxidative damage on bare genomic DNA. Oxidation occurs preferentially at the 5'-guanine of 5'-GG-3' sites, indicative of base damage by DNA-mediated charge transport chemistry. Moreover, oxidative damage occurs at protein-bound sites which are inaccessible to rhodium. Thus, on transcriptionally active DNA within the cell nucleus, DNA-mediated charge transport acts to induce base damage from a distance. Direct interaction of an oxidant is not necessary to generate a base lesion at a specific\r\nsite within the nucleus.
\r\n\r\nAll of these observations indicate that charges can migrate along DNA within the cell. These observations require a reconsideration of cellular mechanisms for DNA damage and repair, and present new avenues for exploration in the design of DNA-based drugs and therapies.
", "doi": "10.7907/ZQ5T-9Z15", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:8157", "collection": "thesis", "collection_id": "8157", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03212014-095851500", "primary_object_url": { "basename": "odom 2001.pdf", "content": "final", "filesize": 39129217, "license": "other", "mime_type": "application/pdf", "url": "/8157/1/odom 2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Application of Metallointercalators in Recognition of and Charge Transport in Nucleic Acids", "author": [ { "family_name": "Odom", "given_name": "Duncan T.", "orcid": "0000-0001-6201-5599", "clpid": "Odom-Duncan-T" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Metal complexes that utilize the 9,10-phenanthrene quinone diimine (phi) moiety\r\nbind to DNA through the major groove. These metallointercalators can recognize DNA\r\nsites and perform reactions on DNA as a substrate. The site-specific metallointercalator\r\n\u039b-1-Rh(MGP)_2phi^(5+) competitively disrupts the major groove binding of a transcription\r\nfactor, yAP-1, from an oligonucleotide that contains a common binding site. The\r\ndemonstration that metal complexes can prevent transcription factor binding to DNA site-specifically\r\nis an important step in using metallointercalators as therapeutics.
\r\n\r\nThe distinctive photochemistry of metallointercalators can also be applied to\r\npromote long range charge transport in DNA. Experiments using duplexes with regions\r\n4 to 10 nucleotides long containing strictly adenine and thymine sequences of varying\r\norder showed that radical migration is more dependent on the sequence of bases, and\r\nless dependent on the distance between the guanine doublets. This result suggests that\r\nmechanistic proposals of long range charge transport must involve all the bases.
\r\n\r\nRNA/DNA hybrids show charge migration to guanines from a remote site, thus\r\ndemonstrating that nucleic acid stacking other than B-form can serve as a radical bridge.\r\nDouble crossover DNA assemblies also provide a medium for charge transport at\r\ndistances up to 100 \u00c5 from the site of radical introduction by a tethered metal complex.\r\nThis radical migration was found to be robust to mismatches, and limited to individual,\r\nelectronically distinct base stacks. In single DNA crossover assemblies, which have\r\nconsiderably greater flexibility, charge migration proceeds to both base stacks due to\r\nconformational isomers not present in the rigid and tightly annealed double crossovers.
\r\n\r\nFinally, a rapid, efficient, gel-based technique was developed to investigate\r\nthymine dimer repair. Two oligonucleotides, one radioactively labeled, are photoligated\r\nvia the bases of a thymine-thymine interface; reversal of this ligation is easily visualized\r\nby gel electrophoresis. This assay was used to show that the repair of thymine dimers\r\nfrom a distance through DNA charge transport can be accomplished with different\r\nphotooxidants.
\r\n\r\nThus, nucleic acids that support long range charge transport have been shown to\r\ninclude A-track DNA, RNA/DNA hybrids, and single and double crossovers, and a\r\nmethod for thymine dimer repair detection using charge transport was developed.\r\nThese observations underscore and extend the remarkable finding that DNA can serve a\r\nmedium for charge transport via the heteroaromatic base stack.
\r\n\r\n", "doi": "10.7907/yz1f-w961", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:4085", "collection": "thesis", "collection_id": "4085", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10142005-111510", "primary_object_url": { "basename": "Kelley_so_1999.pdf", "content": "final", "filesize": 13703858, "license": "other", "mime_type": "application/pdf", "url": "/4085/1/Kelley_so_1999.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Electron transfer through the DNA double helix: spectroscopic and electrochemical studies", "author": [ { "family_name": "Kelley", "given_name": "Shana O.", "orcid": "0000-0003-3360-5359", "clpid": "Kelley-S-O" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Unknown", "given_name": "Unknown" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nThe DNA helix, containing a stacked array of aromatic base pairs, presents a novel medium in which electron transfer mediated by a molecular [pi]-stack can be investigated. To probe electron transfer through DNA, we have constructed duplex assemblies modified with photo- and redox-active probes and applied spectroscopic and electrochemical approaches to the study of DNA-mediated charge transport.\r\n\r\nPhotoinduced electron transfer between intercalators was examined as a function of distance in a series of small DNA duplexes covalently modified with ethidium (Et) and [...]. At distances up to 35 [...], electron transfer occurs on the subnanosecond time scale ([...]). In duplexes containing disruptive base mismatches, large decreases in electron-transfer yields are observed, confirming that the electron transfer pathway proceeds through the stacked base pairs. Hence, it was demonstrated for the first time that DNA-mediated electron transfer between intercalators is exceptionally efficient, only weakly dependent on distance, but highly sensitive to perturbations in base stacking.\r\n\r\nTo investigate a DNA base within the [pi]-stack as a reactant, ethidium-modified duplexes containing the base analogue deazaguanine were synthesized. The photooxidation of deazaguanine by ethidium also proceeds on a subnanosecond time scale ([...]) and exhibits a shallow distance dependence. The efficiency and overall distance dependence is sensitive to the stacking of deazaguanine as determined by flanking sequence. These studies again showed that the DNA base stack can mediate extremely fast, long-range charge transport, and further elucidated that stacking interactions are critical in modulating the efficiency of this phenomenon.\r\n\r\nUsing base-base photochemistry, electron transfer through DNA was probed directly without external donors and acceptors. Using fluorescent analogues of adenine that selectively oxidize guanine, electron transfer through the DNA [pi]-stack was investigated as a function of reactant stacking and energetics. Small variations in each of these factors lead to remarkable changes in the kinetics of DNA-mediated electron transfer and values of [beta], a parameter reflecting the exponential dependence of electron transfer on distance, were measured ranging from [...] to [...]. The DNA base stack was shown to exhibit insulator to \"wire\"-like properties, depending on the structure and energetics of reactants employed to probe this medium.\r\n\r\nTo investigate DNA-mediated electron transfer using electrochemical methods, we assembled DNA films and incorporated intercalating redox-active molecules into the monolayers. Surface characterization techniques were employed to determine the orientation of the DNA helices within the films. With the intercalator daunomycin crosslinked to DNA duplexes immobilized on gold, efficient electron transfer over distances greater than 30 [...] was observed. Base mismatches also attenuate this long-range reaction, providing a new method for the electrochemical detection of genomic mutations.\r\n\r\nThese studies have provided essential measurements of electron transfer in DNA over known, fixed distances. It is now apparent that stacking interactions modulate the efficiency of this phenomenon, an observation that may explain the range of conflicting results reported within this field. Moreover, as experimental evidence increasingly supports the notion that ultrafast charge transport can occur through the DNA helix over long distances, the implications for biological systems can now be considered. Our findings point to the DNA [pi]-stack as not only a carrier of genetic information, but also a pathway which is conducive to charge transport.\r\n", "doi": "10.7907/HZ7H-AD50", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:17792", "collection": "thesis", "collection_id": "17792", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12102025-233557591", "primary_object_url": { "basename": "Lim_AC_1998.pdf", "content": "final", "filesize": 60742266, "license": "other", "mime_type": "application/pdf", "url": "/17792/1/Lim_AC_1998.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Investigation of RNA Tertiary Structure and Function by Transition Metal Complexes", "author": [ { "family_name": "Lim", "given_name": "Ai Ching", "clpid": "Lim-Ai-Ching" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Phenanthrenequinone diimine (phi) complexes of rhodium(III) were employed to\r\nprobe RNA secondary and tertiary structure. These complexes bind via intercalation in\r\nopen major grooves of RNA and upon irradiation promote strand scission. By probing\r\nboth synthetic and natural molecules containing a variety of tertiary motifs, a systematic and\r\npredictive understanding of the factors involved in RNA recognition by these complexes is\r\nsought. The metal complex Rh(phen)2phi3+ (phen = 1,10-phenanthroline) recognizes and\r\ncleaves synthetic triple helices selectively over double helices. The cleavage sites are\r\ndependent upon maximizing overlap between the phi ligand and the basepairs, and\r\nminimizing charge repulsion between the metal complex and protonated bases. These\r\ncleavage sites have proven useful in explaining rhodium complex cleavage in natural\r\nsystems such as tRNAPhe. With these complexes, we also seek to investigate the\r\ndifferences and similarities in RNA and DNA secondary and tertiary folding, by probing\r\nthe tertiary structure of tDNAPhe compared to tRNAPhe. These complexes have elucidated\r\nthe B-form nature of the DNA duplex as well as the tertiary folding of the DNA molecule,\r\nthus shedding light on the feasibility of using DNA analogs of RNA for structural studies.\r\nThese shape selective probes have also been applied to probe the tertiary structure of HIV\r\nand BIV (TAR (trans-activation response) RNAs. \u0394-Rh(phen)2phi3+ binds with high\r\naffinity (Kb= 6.1 \u00b1 1.3 x 105 M-1) and specificity to sites at and across from a bulge\r\nregion which is the recognition element for the binding of the Tat (trans-activating) peptide.\r\nImportantly, the metal complex recognizes an RNA base-triple the formation of which is\r\nnecessary for transactivation. Derivatives of Rh(phen)2phi3+, Rh(MGP)2phi5+(MGP = 4-\r\nguanidylmethy 1-1, 10-phenanthroline) and Rh(GEB)2phi5+ (GEB = 4-(2-guanidylethyl)-4'methy\r\n1-2,2'-bipyridine) where guanidinium moieties have been added to the ancillary\r\nligands of the rhodium complex, show enhanced affinity and selectivity for HIV and BIV\r\nRNA sequences. This is due to the guanidinium moieties mimicking the arginine side\r\nchains on the native Tat peptide, and making non-specific contacts with the phosphate\r\nbackbone of the RNA. However, even without these functionalities, shape-selection,\r\nmatching the shape of the small metal complex to its nucleic acid target, provides sufficient\r\nselective stabilization for RNA site discrimination. Indeed, these complexes compete\r\neffectively with the specific Tat peptides for their binding sites on their respective TAR\r\nRNAs. These complexes therefore employ shape selection to recognize structural\r\nvariations along the RNA polymer which are important for protein recognition. Shape-selective\r\nrecognition could also be applied to the design of novel small molecules to target\r\nnucleic acid sites with high site-selectivity, in the development of molecules to inhibit\r\nprotein recognition, and, potentially, in the design of new chemotherapeutics.", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:17590", "collection": "thesis", "collection_id": "17590", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08042025-205701232", "primary_object_url": { "basename": "Johann_TW_1997.pdf", "content": "final", "filesize": 76248947, "license": "other", "mime_type": "application/pdf", "url": "/17590/1/Johann_TW_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Sequence-Specific Inhibition of DNA Polymerase by Phenanthrene Quinone Diimine Complexes of Rhodium(III)", "author": [ { "family_name": "Johann", "given_name": "Timothy Wilmot", "orcid": "0000-0003-2212-9684", "clpid": "Johann-Timothy-Wilmot" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The DNA binding characteristics of several phenanthrenequinone\r\ndiimine (phi) complexes of rhodium (III) as well as their ability to inhibit\r\nfunctionally DNA polymerase have been investigated. Affinity constants\r\nhave been determined to be 5x107 M-1 and 1x108 M-1 for \u0394 and \u039b 1Rh(\r\nMGP)2phi5+ binding to the DNA sequences 5'-CATCTG-3' and 5'-\r\nCATATG-3' respectively. The exchange rate, at 21\u00b0C, has been determined to\r\nbe 16 s-1 for the binding of 1-\u039b-Rh(MGP)2phi5+ to 5'-CATATG-3' through the\r\nuse of variable temperature 1H-NMR. Similar 1H-NMR experiments were\r\ncarried out to determine the kinetics of the interaction of 1-\u0394-Rh(MGP)2phi5+\r\nwith a duplex DNA of the sequence 5'-CGCATCTGAC-3'. 1-\u039b-Rh(\r\nMGP)2phi5+, 1-\u0394-Rh(MGP)2phi5+, and Rh(MT)phi3+, which binds to 5'-\r\nTGCA-3', were found to be potent sequence-specific inhibitors of DNA\r\npolymerase. All of these complexes bind to DNA through intercalation. In\r\nexperiments where two templates competed for extension by DNA\r\npolymerase, these complexes were shown to inhibit the extension of\r\ntemplates containing their binding sequences as compared to control\r\ntemplates. Furthermore, in direct competition experiments containing two\r\ntemplates, where each contained a binding sequence for a different metal\r\ncomplex, the relative activity of DNA polymerase on each template was\r\n\"tuned\" by the addition of metal complex specific for that template. \u0394-Rh(\r\nDPB)2phi3+ was also found to be a potent inhibitor of DNA polymerase,\r\nbut not in a template-specific manner. The relative potency of sequence- specific\r\ninhibition shown by 1-\u039b-Rh(MGP)2phi5+, 1-\u039b-Rh(MGP)2phi5+, and\r\nRh(MT)2phi3+ was compared to the binding kinetics, complex size, complex\r\ncharge, binding affinity and binding induced DNA distortion for these\r\ncomplexes. Greater DNA distortion was found to correlate with greater\r\ninhibition. These studies have shown that these molecules not only bind to\r\nDNA in a sequence-specific manner, but can functionally inhibit enzymatic\r\nreactions in a sequence-specific manner as well.", "doi": "10.7907/fb9e-rw88", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17615", "collection": "thesis", "collection_id": "17615", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08112025-225322656", "primary_object_url": { "basename": "Arkin_MR_1997.pdf", "content": "final", "filesize": 88219433, "license": "other", "mime_type": "application/pdf", "url": "/17615/1/Arkin_MR_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Investigations of DNA-Mediated Electron Transfer Reactions with Metallointercalators", "author": [ { "family_name": "Arkin", "given_name": "Michelle R.", "orcid": "0000-0002-9366-6770", "clpid": "Arkin-Michelle-R" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Marcus", "given_name": "Rudolph A.", "orcid": "0000-0001-6547-1469", "clpid": "Marcus-R-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The array of \u03c0-stacked base pairs in DNA represents a novel medium for electron transfer\r\nreactions, and metallointercalators have served as useful tools to study this chemistry. Ultrafast\r\nkinetic measurements indicate that photoinduced electron transfer reactions between\r\nM(phen)2(X2dppz)2+ (M = Ru, Os; dppz = dipyrido[3,2-a:2,3-c]phenazine; X = H, CH3) [M(II)]\r\nand Rh(phi)2bpy3+ (phi = phenanthrenequinone diimine) [Rh(III)] can occur with rates> 3 x 1010\r\ns-1. Recombination reactions between M(III) and Rh(II) are also very fast (~ 1010 s-1), and rates\r\nare found to be independent of the loading of \u0394-Rh(phi)2bpy3+ on DNA. However, reaction rates\r\nand efficiencies are highly sensitive to i) the structure and chirality of intercalators and ii) the\r\nsequence and conformation of the DNA double helix. Photoinduced reactions between Ru(II) and\r\nRh(III) bound to the DNA helix and to SOS micelles, which lack the ordered \u03c0-stacked array, are\r\nalso compared. In contrast to DNA, quenching in micelles occurs by diffusion. The details of\r\nintercalation and DNA sequence are thus found to be important characteristics of DNA-mediated\r\nET reactions.
\r\n\r\nTo study long-range reactions through DNA, metallointercalator-DNA conjugates have\r\nbeen prepared. Rh(III) and novel trisheteroleptic complexes of Ru(II) are tethered to the 5'-termini\r\nof oligonucleotides by solid- and solution-phase methods, and these complexes have provided\r\nspectroscopic and photochemical tools to characterize chimeric structures. In addition to\r\nexperiments in which DNA serves as a molecular bridge connecting donor and acceptor, the double\r\nhelix may also serve as a reactant in electron transfer chemistry. Ru(III) oxidants have been\r\ngenerated in situ by a flash-quench methodology and have been found, by transient absorption\r\nspectroscopy, to oxidize G residues in DNA. Furthermore, using a tethered Ru(III)-DNA\r\nconjugate, oxidation products are observed 37 \u00c5 from the metallointercalator. These investigations\r\nof DNA-mediated electron transfer reactions contribute to our understanding of oxidative damage\r\nin DNA and may lead to a novel class of DNA-based biosensors.
", "doi": "10.7907/4ggg-6x62", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17541", "collection": "thesis", "collection_id": "17541", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07182025-180545892", "primary_object_url": { "basename": "Lin_SC_1997.pdf", "content": "final", "filesize": 38508661, "license": "other", "mime_type": "application/pdf", "url": "/17541/1/Lin_SC_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Molecular Recognition of DNA by Rhodium(III)-Zinc Finger Peptide Chimeras", "author": [ { "family_name": "Lin", "given_name": "Susanne Chosein", "clpid": "Lin-Susanne-Chosein" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Covalent chimeras of zinc finger peptide domains with\r\nphenanthrenequinone diimine (phi) complexes of rhodium (III) have been\r\ndesigned, synthesized and their DNA recognition characteristics examined. The\r\nrhodium complex binds in the major groove of DNA by intercalation and allows\r\nthe attached peptide to interact with DNA in a sequence-specific manner.\r\nChimeras of [Rh(phi)2(bpy')]3+ (bpy' = 4-(4-carboxybutyl), 4'-methyl-2,2'bipyridine)\r\nand [Rh(phi)2(phen')]3+ (phen' = (5-amidoglutaryl)-1,10-phen-anthroline)\r\nand four different zinc finger peptides (Sp1 finger 2 and 3, ADR1b and\r\nADR1b-Ala) have been successfully synthesized using solid phase coupling\r\nmethodology. Electronic spectroscopy showed the rhodium complex and\r\npeptide to be essentially independent units. A method to successfully fold the\r\npeptide portion of the chimera with zinc has been developed, and 1H HMR\r\nspectroscopy has been used to confirm folding. The resultant chimeras bind\r\ntightly to DNA, and the rhodium intercalator promotes DNA cleavage with\r\nphotoactivation. Analysis of the DNA sites targeted by the chimeras on DNA\r\nrestriction fragments have demonstrated that the peptide can direct new\r\nrecognition. Variations in the rhodium complexes and peptides resulted in\r\ndifferences in specificity as seen by photocleavage. Studies on smaller\r\noligonucleotides containing the recognition sequences have shown the rhodium -\r\nSp1-2 chimera to bind with affinities of 107-108 M-1 for its target sites. Hence,\r\nformation of rhodium(III) - zinc finger chimeras provide a route to establish\r\nhigh affinity DNA binding by a single zinc finger domain. At some sites, the\r\nrhodium complex and zinc finger appeared to bind independently to adjacent\r\nsegments. For the [Rh(phi)2(phen')]3+ - Sp1 - 2 chimeras, a strong high affinity\r\nsite (Ka greater than or equal to 108 M-1) was observed, where it was postulated that the rhodium\r\ncomplex and zinc finger bind to the opposite strands of the GCG binding site in a\r\ncooperative fashion. These rhodium (III) - zinc finger chimeras represent a new\r\nroute to examine the specific interactions of a single zinc finger with DNA in\r\nchemical detail and provide the basis to build a family of sequence-specific DNA\r\nbinding molecules.", "doi": "10.7907/mss5-wf97", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:11902", "collection": "thesis", "collection_id": "11902", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11082019-094749072", "primary_object_url": { "basename": "jenkins-y-1996.pdf", "content": "final", "filesize": 6480454, "license": "other", "mime_type": "application/pdf", "url": "/11902/1/jenkins-y-1996.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Dipyridophenazine Complexes of Ruthenium(II) as Luminescent Reporters of DNA", "author": [ { "family_name": "Jenkins", "given_name": "Yonchu", "clpid": "Jenkins-Yonchu" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Imperiali", "given_name": "Barbara", "clpid": "Imperiali-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The search for novel diagnostics for DNA detection has generated interest in the potential applications of polypyridyl complexes of ruthenium(II). Recently, it was reported that dipyridophenazine complexes of ruthenium(II) may serve as molecular light switches for DNA. These complexes, which bind DNA avidly through intercalation, show no luminescence in aqueous solutions. Upon intercalation into double-helical DNA and the concomitant protection of the phenazine ring from quenching by interaction with water, intense photoluminescence is apparent. The light switch effect as a function of nucleic acid sequence and conformation was examined for [Ru(phen)2dppz]2+ and [Ru(bpy)2dppz]2+. The emission properties of these complexes were found to be extremely sensitive to the nature of the intercalation environment with strong correlations between the luminescence parameters and the level of water protection afforded by the double helix. In order to impart sequence specificity to the light switch effect, various methods have been developed for appending a functionalized [Ru(phen)2dppz]2+ to the 5' terminus of oligonucleotides, both on the solid support and in solution. Assays for analyzing the structural integrity of the resulting conjugates are described. These ruthenated oligonucleotides can serve as enzyme substrates, enabling the construction of long metalated oligonucleotides not easily prepared using chemical synthesis. In order to evaluate their utility as useful DNA diagnostics, a series of ruthenated oligonucleotides were synthesized and their photophysical properties characterized. Biochemical analysis of oligonucleotide duplexes containing ruthenated strands showed no significant structural perturbation of the duplex as a result of the ruthenium modification. The overall results of this investigation suggest that an oligonucleotide functionalized with a dppz complex of ruthenium may be used to target single-stranded DNA in a sequence-specific fashion and that this derivative could be extremely valuable in the development of novel hybridization probes for both heterogeneous and homogeneous assays.
", "doi": "10.7907/n4jx-hp63", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:17681", "collection": "thesis", "collection_id": "17681", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09152025-212928780", "primary_object_url": { "basename": "Terbrueggen_R_1996.pdf", "content": "final", "filesize": 65415187, "license": "other", "mime_type": "application/pdf", "url": "/17681/1/Terbrueggen_R_1996.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Exploring the Direct and Indirect Readout of DNA with Phenanthrenequinone Diimine Complexes of Rhodium(III)", "author": [ { "family_name": "Terbrueggen", "given_name": "Robert Henry", "clpid": "Terbrueggen-Robert-Henry" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Phenanthrenequinone diimine (phi) complexes of rhodium(III) have been\r\ndesigned and characterized in order to investigate the principles of direct and\r\nindirect readout of double helical DNA The metallointercalator l-Rh(MGP)2phi5+\r\n(MGP = 4-guanidylmethyl-1,10-phenanthroline) binds via intercalation in the\r\nmajor groove of DNA and upon irradiation promotes DNA strand scission. The\r\n\u039b-enantiomer, \u039b-1-Rh(MGP)2phi5+, binds at subnanomolar concentrations to the\r\n6 base pair sequence, 5'-CATATG-3', with enantiospecificity. An essential feature\r\nof this recognition is the sequence-specific unwinding of the DNA helix which\r\npermits direct contacts between guanidinium functionalities on the metal complex\r\nand guanine residues. Deazaguanine substitutions were used to establish direct\r\ncontacts between the N7 nitrogen atoms of guanine and the guanidinium moiety on\r\nthe metal complex. Through an assay developed to test for sequence-specific DNA\r\nunwinding, a 70 \u00b1 10 degrees unwinding of the sequence 5'-CATATG-3' upon\r\nbinding by \u039b-1-Rh(MGP)2phi5+ was established. Thus, the sequence-dependent\r\ntwistability of DNA plays an important role in determining the sequence specificity\r\nof the complex. The \u0394-enantiomer, \u0394-1-Rh(MGP)2phi5+, binds preferentially to\r\nthe 6 base pair sequence, 5'-CATCTG-3'. The hierarchy of recognition sites\r\ndetermined in photocleavage studies on oligonucleotides suggests that DNA\r\nrecognition by this complex also involves sequence specific contacts by the\r\nguanidinium functionalities. Photocleavage studies indicate additional similarities\r\nin the recognition of \u0394 and \u039b-1-Rh(MGP)2phi5+. Both enantiomers of 1-\r\nRh(MGP)2phi5+ display increased binding specificity relative to the parent\r\ncomplex, Rh(phen)2phi3+. The exchange rates of both enantiomers are also\r\ndecreased at least a 1000-fold relative to Rh(phen)2phi3+. Studies in which the\r\nlength of the linker arm between the core of the metal complex and the guanidinium\r\nmoiety was varied demonstrate that proper orientation of the guanidinium moiety is\r\nan essential feature of complex specificity. As the length of the linker arm\r\nincreases, the binding specificity of the complex decreases. DNA recognition\r\nstudies with Rh(APB)2phi5+ (APB= 4-(3-aminopropyl)-4'-2,2'-bipyridine) have\r\ndemonstrated that the amino moiety can also be used to alter the sequence\r\nspecificity of phi complexes of rhodium(III), although the sequence specificity of\r\nthis complex is reduced greatly as compared with \u0394- and \u039b-1-Rh(MGP)2phi5+.\r\nThis work therefore demonstrates that the guanidinium moiety may be used to\r\nenhance both the binding affinity and specificity of phi complexes of rhodium(III).\r\nIn mimicking DNA binding proteins, molecules which recognize their binding sites\r\nthrough direct and indirect readout of the DNA can be designed. Importantly, this\r\nstudy highlights a new structural element of DNA recognition, the sequence-dependent\r\ntwistability of the DNA helix. This sequence-dependent twistability may\r\nbe an essential feature of the recognition of sequences by DNA-binding proteins\r\nand may be powerfully exploited in future design.", "doi": "10.7907/yv86-3710", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:3383", "collection": "thesis", "collection_id": "3383", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09082006-114255", "primary_object_url": { "basename": "Campisi_d_1996.pdf", "content": "final", "filesize": 7329538, "license": "other", "mime_type": "application/pdf", "url": "/3383/1/Campisi_d_1996.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Transition metal complexes as probes of DNA sequence-dependent structure", "author": [ { "family_name": "Campisi", "given_name": "Donna", "clpid": "Campisi-D" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Different transition metal complexes have been applied in probing variations in the structure of double helical DNA. The following probes, which all bind DNA noncovalently, have been utilized: Ru(phen)3(2)+, Ru(TMP)3(2)+, Rh(phen)2phi3+, Rh(TMP)2phi3+, Rh(dmbpy)2phi3+, Ru(phen)2dppz2+, Ru(bpy)2dppz2+, and Rh(bpy)2dppz3+ (phen = 1,10 phenanthroline; TMP = 3,4,7,8,-tetramethyl- 1,10-phenanthroline; phi = 9,10-phenanthrenequinone diimine; dmbpy = 5,5'-dimethylbipyridyl; bpy = bipyridyl; dppz = dipyrido[3,2-a;2',3'-c]phenazine). The local structure recognized by [Delta]-Rh(phen)2phi3+ has been defined by comparisons of photocleavage data on crystallographically characterized oligonucleotides with their structural parameters. A quantitative correlation has been determined between [Delta]-Rh(phen)2phi3+ photocleavage and extent of openness in the major groove due to differential propeller twisting, or interpurine angle. Therefore, [Delta]-Rh(phen)2phi3+ has been developed as a probe of DNA propeller twisting in solution. Differences in reaction pathway partitioning between enantiomers of Rh(phen)2phi3+ are attributed to differing extent of shape complementarity with DNA binding sites. Rh(TMP)2phi3+ has been explored in probing DNA mismatches in solution. Both [Delta]-Rh(phen)2phi3+ and Rh(TMP)2phi3+ sensitively mark local structural perturbations in an oligonucleotide, arising from substitution of a CG base pair with TG and AG mismatches. Rh(phen)2phi3+ and Ru(TMP)3(2) have also been applied in probing structural variations in the context of a long DNA strand. A C7 stretch is targeted by Ru(TMP)3(2), an A DNA probe and Rh(phen)2phi3+, a B DNA probe. These results indicate this sequence is heteronomous, containing wide major and minor grooves. [Delta]- and [Lambda]-Rh(phen)2phi3+ also discriminate structural differences between bent and nonbent DNA fragments. Variations in metal complex-DNA interactions have also been examined by a gel electrophoretic mobility assay. Intercalator size, hydrophobicity of ancillary ligands, metal complex charge, and chirality all influence the extent of DNA retardation. Taken together, these studies demonstrate that transition metal complexes can be profitably and uniquely applied towards exploring DNA structural heterogeneity.", "doi": "10.7907/kay4-sz63", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:4168", "collection": "thesis", "collection_id": "4168", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10182007-103625", "primary_object_url": { "basename": "Pustilnik_mm_1995.pdf", "content": "final", "filesize": 8793470, "license": "other", "mime_type": "application/pdf", "url": "/4168/1/Pustilnik_mm_1995.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The construction and application of metal complexes for DNA strand scission", "author": [ { "family_name": "Pustilnik", "given_name": "Michael Morris", "clpid": "Pustilnik-M-M" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Unknown", "given_name": "Unknown" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nSubstitutionally inert transition metal complexes have been constructed which bind to DNA and deliver additional labile metal ions to promote cleavage of the phosphodiester backbone. Both oxidative and hydrolytic DNA cleavage have been explored through the use of different chelates and reactive metal ions.\n\nTo explore oxidative chemistry, two complexes, [...] and [...], have been synthesized by tethering two bis(2-picolyl)amine or bis(2-ethylpyridyl)amine chelates to the [...] moiety. Both complexes promote site-selective oxidative DNA cleavage in the presence of CuSO4 and a thiol, with the site-selectivity being governed by the [...] core. Copper-ligand binding affinity, complex-DNA binding mode, and the [...]/[...] redox potential all appear to sensitively influence the cleavage efficiency. Product analysis of the DNA cleavage reaction is consistent with hydrogen abstraction from C1', C3', and C4' of the deoxyribose ring. The [...] core therefore serves as a vehicle to deliver oxidative reactions to DNA. These complexes may be valuable for the design of drugs that cleave DNA in vivo.\n\nThe complexes [...], [...], [...], and [...] have also been prepared so as to explore the delivery of secondary metal ions and functionalities to promote DNA hydrolysis. The complexes each have two tethered bis(2-picolyl)amine chelating groups; the latter three complexes also have two 2-dimethylaminoethyl or 3-dimethylaminopropyl groups designed for general acid assistance. DNA hydrolysis has not been established, but [...] promotes efficient DNA cleavage in the presence of [...]. Product analysis of DNA oligonucleotide cleavage by [...] is consistent with a [...] mediated reaction which is remarkably enhanced compared to those of ruthenium complexes lacking the tethered chelates; this reactivity may indicate that [...] promotes abasic site cleavage. [...], which does not sensitize [...] formation, promotes still more efficient DNA cleavage. The mechanism is yet to be established.\n", "doi": "10.7907/h206-6d32", "publication_date": "1995", "thesis_type": "phd", "thesis_year": "1995" }, { "id": "thesis:4213", "collection": "thesis", "collection_id": "4213", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10222007-132632", "primary_object_url": { "basename": "Sardesai_ny_1995.pdf", "content": "final", "filesize": 17895094, "license": "other", "mime_type": "application/pdf", "url": "/4213/1/Sardesai_ny_1995.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Rhodium intercalators as novel peptide delivery systems to the major groove of DNA : towards the design of artificial repressors", "author": [ { "family_name": "Sardesai", "given_name": "Niranjan Y.", "clpid": "Sardesai-N-Y" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nPhenanthrenequinone diimine (phi) complexes of rhodium(III) bearing tethered peptides have been designed to serve as metallointercalating anchors to deliver peptide side chain functionalities for DNA recognition in the major groove. Metal-peptide \ncomplexes containing 11-15 amino acid residues were prepared using two complementary synthetic strategies: by direct coupling of a pendant carboxylate on the coordinatively saturated rhodium complex, [...] (phen'=5-amidoglutaryl-1, 10-phenanthroline), to the N-terminus of a resin-bound peptide in a manner analogous to the chain-elongation step in solid phase peptide synthesis; or by coupling phen' containing the pendant carboxylate to the resin-bound peptide, followed by coordination of [...] to the bidentate chelator attached to the peptide. With coordination complexes which are stable to peptide deprotection and cleavage conditions from the resin, the solid phase synthetic strategies prove convenient to apply. The metal-peptide complexes have been characterized by amino acid analysis, electronic spectroscopy, circular dichroism and mass spectrometry, where a novel pattern of peptide fragmentation facilitates the detailed sequence analysis of the appended peptide. All the metal-peptide complexes bind and, with photoactivation, cleave DNA with evidence of major groove chemistry. Significantly, the DNA site-specificity is seen to depend on the peptide side-chain functional groups. In one series, a single glutamate at position 10 is found to be essential in directing DNA site-recognition to the sequence 5'-CCA-3'. Methylation of the glutamate side chain or single ElOQ, E1OD, E1OA mutations abolish this selectivity. The glutamate is essential to maintain [...]-helicity in the peptide and make base specific contacts, thereby providing a glutamate switch for site-specific DNA recognition. A second series, based on the recognition helix of the phage 434 repressor, reproduces operator binding. Photocleavage and MPE-Fe footprint analysis indicates that these metal-peptide complexes bind to the 5'-ACAA-3' operator sequences as monomers at\n10 nM concentration and differentiate between operator site variants. These studies represent a new strategy to create an array of metal-peptide complexes with differing sequence specificity for DNA and suggest a route to the construction of small molecules that function as artificial repressors.\n", "doi": "10.7907/0v7r-fp28", "publication_date": "1995", "thesis_type": "phd", "thesis_year": "1995" }, { "id": "thesis:4212", "collection": "thesis", "collection_id": "4212", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10222007-131939", "primary_object_url": { "basename": "Shields_tp_1995.pdf", "content": "final", "filesize": 9103344, "license": "other", "mime_type": "application/pdf", "url": "/4212/1/Shields_tp_1995.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "DNA recognition by the enantiomers of Rh(en)2phi3+ : recognition through hydrogen bonding and van der Waals contacts", "author": [ { "family_name": "Shields", "given_name": "Thomas P.", "clpid": "Shields-T-P" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Unknown", "given_name": "Unknown" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nThe enantiomers of Rh(en)2phi[...] (en = ethylene diamine, phi = 9,10-phenanthrene quinone diimine) have been prepared in order to explore systematically the contributions of hydrogen bonding and van der Waals contacts to sequence selective DNA binding. Both enantiomers bind strongly to DNA via intercalation of the aromatic phi ligand, and produce direct strand scission upon irradiation with near-UV light. The cleavage mechanism is consistent with the direct abstraction of the C3'-H atom from the major groove of DNA by a bound, photoexcited Rh(phi)[...] species. Elements of the sequence specificity of [...] and [...] were established by comparing recognition characteristics to those of several [...] complexes containing ancillary saturated amines and a thioether, and by cleavage of oligonucleotide targets containing [...], 7-deazaguanine and deoxyuracil substitutions. Both enantiomers display a strong sensitivity to the inclusion or removal of a single methyl group in the major groove of [...] displays relatively high selectivity in targeting [...] steps and this selectivity is the result of hydrogen bonding between the axial amines of the metal complex and the [...] position of guanine residues. In contrast, [...] is less selective; besides binding to [...] steps, the A-enantiomer also recognizes [...] steps through a positive van der Waals contact between the ancillary methylene groups and the 5-methyl group of thymine. The structural basis for these enantioselective elements of recognition have also been investigated via [...] methods. Both [...] bind to d(GTGCAC)[...] by classical intercalation, and 2D-NOESY studies of the [...] complex demonstrate specific intercalation at the central [...] site. Molecular models show that deep intercalation of the phi ligand ideally locates the ancillary ethylene diamine ligands for the proposed hydrogen bonding and van der Waals contacts. While illustrating the importance of hydrogen bonding and van der Waals contacts in achieving site specificity, the current work also suggests a modular approach to the design of molecules capable of recognizing larger DNA sequences.", "doi": "10.7907/tx6p-mg53", "publication_date": "1995", "thesis_type": "phd", "thesis_year": "1995" }, { "id": "thesis:7740", "collection": "thesis", "collection_id": "7740", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05222013-114326444", "primary_object_url": { "basename": "Lee-I-1994.pdf", "content": "final", "filesize": 32662712, "license": "other", "mime_type": "application/pdf", "url": "/7740/1/Lee-I-1994.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Distinct intron DNA structures in simian virus 40 T-antigen and adenovirus 2 E1A genes", "author": [ { "family_name": "Lee", "given_name": "Inho", "clpid": "Lee-I" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Unknown", "given_name": "Unknown" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Distinct structures delineating the introns of Simian Virus 40 T-antigen and Adenovirus 2 E1A genes have been discovered. The structures, which are centered around the branch points of the genes inserted in supercoiled double-stranded plasmids, are specifically targeted through photoactivated strand cleavage by the metal complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III). The DNA sites that are recognized lack sequence homology but are similar in demarcating functionally important sites on the RNA level. The single-stranded DNA fragments corresponding to the coding strands of the genes were also found to fold into a structure apparently identical to that in the supercoiled genes based on the recognition by the metal complex. Further investigation of different single-stranded\r\nDNA fragments with other structural probes, such as another metal complex bis(1,10-phenanthroline)(phenanthrenequinone diimine)rhodium(III), AMT (4'aminomethyl-4,5',8 trimethylpsoralen), restriction enzyme Mse I, and mung bean\r\nnuclease, showed that the structures require the sequ ences at both ends of the intron plus the flanking sequences but not the middle of the intron. The two ends form\r\nindependent helices which interact with each other to form the global tertiary structures. Both of the intron structures share similarities to the structure of the\r\nHolliday junction, which is also known to be specifically targeted by the former metal complex. These structures may have arisen from early RNA intron structures and may have been used to facilitate the evolution of genes through exon shuffling by acting as target sites for recombinase enzymes.
\r\n", "doi": "10.7907/zhde-t378", "publication_date": "1994", "thesis_type": "phd", "thesis_year": "1994" }, { "id": "thesis:7388", "collection": "thesis", "collection_id": "7388", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01102013-100729852", "primary_object_url": { "basename": "Sitlani-a-1993.pdf", "content": "final", "filesize": 37965882, "license": "other", "mime_type": "application/pdf", "url": "/7388/1/Sitlani-a-1993.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Sequence specific recognition and photocleavage of DNA by phenanthrenequinone diimine complexes of rhodium(III)", "author": [ { "family_name": "Sitlani", "given_name": "Ayesha", "clpid": "Sitlani-A" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Unknown", "given_name": "Unknown" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Sequence-specific recognition and photocleavage of DNA by a series of 9,10- phenanthrenequinone diimine (phi) complexes of rhodium(III) was studied. [Rh(phi)]^(3+) complexes bind to DNA via intercalation of their phi ligand and upon photoactivation promote strand scission. The DNA degradation products formed are consistent with photoreaction of [Rh(phi)]^(3+) intercalated in the major groove of DNA, via abstraction of the C3'-H atom of the deoxyribose. For the complex [Rh(phen)2phi]^(3+) and its derivatives [Rh(X)2phi]^(3+), the primary products are 5' and 3' phosphate termini and nucleic acid bases. For the complex [Rh(phi)2bpy]^(3+) and its derivatives [Rh(phi)2X]^(3+), additional products, dependent on dioxygen concentrations, are characterized as base propenoic\r\nacids and 3'-phosphoglycaldehyde termini. The partitioning between the oxygen dependent and oxygen independent pathways correlates best with how the shapes of these\r\ncomplexes limit access of dioxygen to the C3' deoxyribose position. The shapes of [Rh(phi)]^(3+) complexes also govern their sequence-specific DNA recognition. The more\r\nsterically bulky complexes with methyl or phenyl groups on their ancillary ligands cleave at a subset of sequences recognized by their parent molecules. The \u2206 and \u039b isomers of\r\n[Rh(5,5'-dimethylbpy)2phi]^(3+) cleave specifically at sites that are defined by the consensus sequences 5'-C-T-pu/py-G-3' and 5'-A-C/G-T-C/G-3', respectively. This\r\nsequence-specificity may be understood on the basis of negative steric clashes and positive van der Waals interactions between methyl groups on the metal complex and\r\nthymine methyl groups in the DNA major groove. The complex [Rh(4,4'diphenylbpy) 2phi]^(3+) recognizes the self-complementary eight base-pair sequence 5'CTCTAGAG-\r\n3', both due to its bulky shape and its ability to cooperatively associate through non-covalent dimerization on the DNA helix. [Rh(4,4'-diphenylbpy)2phi]^(3+) is\r\nshown to inhibit sequence-specific cleavage by the restriction enzyme Xbal. It is likely that, like [Rh(phi)]^(3+) complexes, DNA binding proteins exploit shape selection to achieve high levels of sequence-specificity.
\r\n", "doi": "10.7907/phhk-4s21", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:6714", "collection": "thesis", "collection_id": "6714", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10142011-105722078", "primary_object_url": { "basename": "Chow_cs_1992.pdf", "content": "final", "filesize": 7728520, "license": "other", "mime_type": "application/pdf", "url": "/6714/1/Chow_cs_1992.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Transition Metal Complexes as Probes for Higher-Order Structure in RNA", "author": [ { "family_name": "Chow", "given_name": "Christine S.", "clpid": "Chow-Christine-S" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Unknown", "given_name": "Unknown" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A series of transition metal complexes were employed to examine higher-order structure in ribonucleic acids. Our results indicate that the complexes Ru(phen)_3^(2+), Ru(TMP)_3^(2+), Rh(TMP)_3^(3+), Rh(phen)_2phi^(3+), Rh(phi)_2bpy^(3+), and Rh(DIP)_3^(3+) (phen = 1,10-phenanthroline; TMP = 3,4,7,8,-tetramethy1-1,10-phenanthroline; phi = 9,10- phenanthrenequinone diimine; bpy = bipyridyl; DIP = 4,7-dipheny1-1,10-phenanthroline) have different affinities for tRNA and bind RNA by several different modes of interaction, as shown through a variety of biophysical analyses. These differences in binding have been attributed to the different shapes of the metal complexes. Photolysis of the metal complexes promotes cleavage of native, structured RNA at diverse and novel sites with comparable efficiency and analogous product formation as found with cleavage of double-stranded DNA. As on DNA, RNA strand scission promoted by the complexes of rhodium(III) occurs through an oxidative pathway with the sugar moiety as the target. Reactions with the complexes of ruthenium(II) are consistent with mediation by singlet oxygen with the nucleic acid base as the target. The site selectivity associated with cleavage appears to be based upon the different binding properties and therefore the molecular shapes of the complexes. Ru(TMP)_3^(3+) cleaves at a subset of solvent accessible sites cleaved by Ru(phen)_3^(2+). Different sites of cleavage on tRNA are apparent with the rhodium complexes, Rh(phen)_2phi^(3+), Rh(phi)_2bpy^(3+), and Rh(DIP)_3^(3+), while Rh(TMP)_3^(3+) does not promote strand scission of RNA. In particular, Rh(phen)_2phi^(3+) targets sites of triple-base interaction, D-T\u03a8C loop interactions, and helix-loop junctions in tRNA, where the major groove is open and accessible. Rh(DIP)_3^(3+) targets RNA loop structures and G-U mismatches that occur within an RNA double-helix. These shape-selective probes, which promote strand scission of tRNA at unique sites, have also been applied to probe mutant tRNAs and to delineate the structure of 5S rRNA. This study demonstrates that small molecules can recognize distinct structures along an RNA strand and suggests that these structures may be utilized for specific recognition by proteins.", "doi": "10.7907/npv2-kv27", "publication_date": "1992", "thesis_type": "phd", "thesis_year": "1992" } ]