Liquid-liquid phase separation (LLPS) is a thermodynamic process by which a mixture of solutions de-mixes into separate, coexisting phases. In cells, macromolecules such as proteins and nucleic acids can undergo LLPS to form membraneless structures known as biomolecular condensates. These condensates are usually liquid-like droplets highly concentrated in the species they are composed of. Condensates are crucial to many cellular processes and functions, such as protein assembly, gene regulation, subcellular organization, storage, and stress response. On a larger scale, condensates are also implicated in various neurodegenerative diseases, such as Huntington's and Alzheimer's disease.
\r\n\r\nDNA condensates are easy to engineer due to the programmable nature of the molecule. DNA nanostars are multi-armed junctions with double-stranded arms and typically single-stranded, palindromic overhangs or 'sticky ends' that allow for transient interactions between the molecules. They can phase-separate to form liquid-like droplets at the microscopic scale. Centrifugation of a high concentration solution of nanostars coalesces condensates into a macroscopic liquid that is visible to the naked eye.
\r\n\r\nIn biology, one of the key functions of condensates is to act as compartments and localize various molecules, such as enzymes and nucleic acids. Our goal is to engineer macroscopic DNA liquids to similarly act as compartments that can separate multiple biomolecular targets. We propose that these macroscopic DNA liquids are a potentially novel method for multiplexed separation that is fast, simple to use, and biocompatible with various high-value targets, such as protein therapeutics.
\r\n\r\nWe designed multiple immiscible DNA liquids of different densities to act as macroscopic compartments. We present a set of design principles for engineering nanostars to create liquid layers in a microcentrifuge tube. We show via UV absorbance measurements how various nanostar features, such as arm length, sticky end strength, and valency, affect liquid phase density, which determines the order of layering between liquids. We further show that the interface quality between pairwise liquids is determined by a combination of density differences between liquids and the orthogonality of nanostar sticky ends, and we devise a metric to calculate this orthogonality. Using these design principles, we are currently able to create up to five orthogonal liquid layers in a tube.
\r\n\r\nWith these DNA liquid layers, we envision separation to be a two step process. The first step is to localize specific target biomolecules into these layers. We demonstrate localization of oligos in a multilayer DNA liquid system by modifying nanostars with tag regions complementary to the target strand. We also demonstrate localization of fluorescent streptavidin to a single DNA liquid layer by modifying nanostars with a streptavidin-binding aptamer. The second step is to release targets from the liquid layers so that they can be collected for downstream use. After localization of the aforementioned targets, we added strands complementary to either the tag region or the aptamer. This causes targets to be displaced from their binding moiety and released into the supernatant.
", "doi": "10.7907/3639-5v91", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17203", "collection": "thesis", "collection_id": "17203", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05052025-211734908", "type": "thesis", "title": "Enriching Architectures for Biosensing and Motor-Filament Systems Through the Programmability of DNA", "author": [ { "family_name": "Guareschi", "given_name": "Matteo Michele", "orcid": "0000-0002-5197-3158", "clpid": "Guareschi-Matteo-Michele" } ], "thesis_advisor": [ { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Since its inception, the field of DNA nanotechnology has focused on studying the fundamental behaviors and capabilities of engineered nucleic acids. A deep understanding of this toolkit has enabled advancements in several fields, for research tools and in translational applications. Together with its programmability and nanometric resolution, the great promise of DNA nanotechnology lies in the incorporation of structure and function in a single molecule. In this work, we show how these advantages can be leveraged to expand the capabilities of two different systems: a sensor for biomarkers and a motor-filament architecture. During our exploration, we also discover and work to overcome some of the less obvious limitations of the technology, shining light on more foundational questions.
\r\n\r\nWe demonstrate an electrochemical biosensor based on a DNA origami that can detect and quantify nucleic acids and proteins in a package easily adaptable to different analytes by simply replacing the binder molecules. Upon target binding, the structure undergoes a large conformational change, bringing a multitude of redox reporters to the electrode surface where an electric current can be measured. The high number of reporter molecules on a single detector results in improved signal gain per binding event, allowing for the detection of low analyte concentrations, while the conformational change yields an unprecedented gain between the off and on state. We demonstrate how the system can be readily adapted to different analyte molecules and reused over several cycles to analyze multiple samples. We then run simulations of the detector molecule to understand structural deformations intrinsic to this design, in order to optimize the number and placement of the redox reporters. We discover and investigate a phenomenon that causes significant curling of the DNA origami, possibly limiting the contribution of many of the reporter molecules. We explore experimental directions to mitigate the issue by changing the configuration of the redox molecules and by designing stiffer sensors.
\r\n\r\nWe then set out to integrate DNA origami-based nanostructures with an engineered dynein protein that can bind to and kick double-stranded DNA instead of tubulin. Motor-filament architectures have been studied as the main mechanism for cellular transport and as a system that can exhibit mesoscopic active matter behaviors in biology, but the relative difficulty of engineering microtubules has hindered the exploration of their properties. The high-resolution programmability of DNA nanostructures makes them prime candidates to overcome this obstacle and this study has been enabled by the recent development of new protein motors where the tubulin binding domain is replaced by a DNA binding domain. We first look at DNA nanotubes, structures that resemble microtubules, but that retain a level of programmability that is typical of DNA nanotechnology. By exploiting the DNA strand displacement technique, we incorporate machinery that enables new behaviors, with a focus on different ways to turn gliding on and off by stopping the DNA nanotubes.
\r\n\r\nWe then turn our focus to more complex gliders designed with DNA origami. We explore the space of DNA origami polymers in order to assemble superstructures that can be detected under light microscopy, encountering again issues of deformations due to the addition of overhangs. We then assess the gliding capabilities of DNA origami, designing ways to incorporate motor binding sequences on them, but we find that DNA origami sticks nonspecifically to the engineered dynein motors. After testing several different hypotheses, we gather evidence that this interaction might be caused by the large sequence variability of the scaffold strand in DNA origami, coupled with the recognition of spurious binding sequences by the motor proteins.
", "doi": "10.7907/fmhp-r892", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17284", "collection": "thesis", "collection_id": "17284", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282025-171526879", "type": "thesis", "title": "Construction of Long, Complex, and Diverse DNA Sequences", "author": [ { "family_name": "Robinson", "given_name": "Noah Evan", "orcid": "0009-0000-2481-9596", "clpid": "Robinson-Noah-Evan" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Joseph", "orcid": "0000-0001-9598-2454", "clpid": "Parker-J" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The DNA molecule encodes the information required for biological systems to carry out a broad range of functions. The understanding of this relationship has sparked inquiries across vast fields of biology and biological engineering as we read, edit, and write the genetic information of organisms. Great advancements have been made toward these pursuits, from revolutions in DNA reading with long read sequencing and the ability to generate terabytes of data from a single run to the breakthroughs in DNA editing with the major advancements in CRISPR/Cas technologies over the last decade. However, writing DNA, as the ability to construct DNA of any length, complexity, or diversity, lags significantly behind our capacity for reading and editing.
\r\n\r\nDNA oligo synthesis can only reach short lengths of a few hundred nucleotides of single stranded DNA. The field of DNA assembly develops the methods for stitching together DNA oligos and DNA fragments into larger constructs. The current field applies a broad range of approaches that each occupy their own niche due to their unique set of advantages and disadvantages. No existing technique is able to assemble a large number of DNA fragments simultaneously with high accuracy and without placing restrictions on the sequences being assembled. This is because all existing DNA assembly technologies rely on the information contained within the complementary sequences of the DNA molecules being constructed to direct the assembly.
\r\n\r\nTo meet the demand for robust DNA assembly, we have developed a new DNA assembly technique named Sidewinder which separates the information that guides assembly from the final assembled sequence using DNA 3-Way junctions. We demonstrate the transformative nature of the Sidewinder technique with highly robust and accurate assembly of complex DNA sequences of both high GC and high repeats, a 40-piece multi-fragment assembly, a parallel construction of multiple distinct genes in the same reaction, and construction of a combinatorial library with a large number of diversified positions across the entire length of the gene for high coverage of a library of 442,368 variants.
\r\n\r\nWhere Sidewinder excels at the assembly of oligos to the kilobase scale, we have made a series of advancements to an existing 2-Way junction assembly technique, USER cloning, for the accurate and efficient assembly of PCR-based DNA inputs. We characterize these improvements with a series of assemblies where we achieve an average accuracy over 95%, gain 3 orders of magnitude increase in yield of transformants, and conduct large multi-fragment assemblies beyond what was previously possible with the technique. We then interface these two state-of-the-art capacities for the rapid and efficient construction of a complex 10 kilobase sequence de novo and entirely cell-free.
", "doi": "10.7907/qtq1-dv04", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17148", "collection": "thesis", "collection_id": "17148", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04092025-234838672", "primary_object_url": { "basename": "DavidsonSamuel2025_Thesis (Version 2025-04-08).pdf", "content": "final", "filesize": 24258588, "license": "cc_by_nc_sa", "mime_type": "application/pdf", "url": "/17148/1/DavidsonSamuel2025_Thesis (Version 2025-04-08).pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Localized Catalytic DNA Circuits for Integrated Information Processing in Molecular Machines", "author": [ { "family_name": "Davidson", "given_name": "Samuel Ryan", "orcid": "0000-0002-8081-3591", "clpid": "Davidson-Samuel-Ryan" } ], "thesis_advisor": [ { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "thesis_committee": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "This thesis supports the long-term goal of engineering molecular devices with computational complexity akin to cells. Like cells, artificial molecular devices can benefit from integrating multiple computational modalities.
\r\n \r\nTo that end, this thesis advances molecular computing systems in three modalities: dynamic molecular assembly, well-mixed circuits, and spatially-organized cascades. Specifically, it introduces methods to enhance control over DNA structural assembly, well-mixed DNA circuits, and DNA circuits localized to a DNA origami surface.
\r\n \r\nAs DNA structural assembly grows increasingly complex, so too grows the potential for off-target structures. This issue can be addressed through developmental self-assembly, where components join a growing structure in a programmed sequence under controlled kinetics. The scope of developmental self-assembly is here expanded by a method enabling specific pathway selection among multiple encoded options.
\r\n \r\nWell-mixed DNA circuits require catalytic motifs for signal restoration and amplification. A catalytic motif is presented where two input strands cooperate to control catalysis. This motif could enhance AND gates and thresholding, and could enable adaptive memories and learning behaviors in DNA-based neural networks.
\r\n\r\nLocalized DNA circuits lack cascadable catalytic mechanisms for signal restoration and amplification. Two designs for a localized catalytic mechanism are presented. Each omits any intermediate diffusible species to support nanodevices compatible with uncontrolled environments, as in biomedical contexts. This constraint leads to design lessons; principally, we respond to leak in the first design through geometric constraints in the second design.
", "doi": "10.7907/831v-vq95", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16363", "collection": "thesis", "collection_id": "16363", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04292024-172707491", "primary_object_url": { "basename": "Thesis_Cherry_Kevin_Redacted.pdf", "content": "final", "filesize": 20088636, "license": "other", "mime_type": "application/pdf", "url": "/16363/6/Thesis_Cherry_Kevin_Redacted.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Molecular Pattern Recognition and Supervised Learning in DNA-Based Neural Networks", "author": [ { "family_name": "Cherry", "given_name": "Kevin Matthew", "orcid": "0000-0002-2343-0754", "clpid": "Cherry-Kevin-Matthew" } ], "thesis_advisor": [ { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Yue", "given_name": "Yisong", "orcid": "0000-0001-9127-1989", "clpid": "Yue-Yisong" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Adaptation in nature begins at the subcellular, molecular level with the delicate interplay of biomolecule cascades orchestrating the myriad function of cells. The intermingling activity of these cells becomes expressions of complex behavior of multi-cellular system. Nature provides a dazzling array of examples showcasing the variations of intelligent functions. However, in the realm of synthetic construction, what systems have humans managed to engineer, and what are the boundaries of our technological power? In comparison to nature's repertoire, mankind's accomplishments appear rather modest. The intricate behaviors observed in intelligent organisms emerge from the collective interactions and feedback loops among their constituent elements, resulting in the emergence of novel properties and phenomena. To develop large-scale engineered systems exhibiting ever more brain-like, intelligent behaviors, we must first devise new molecular architectures and algorithms designed for adaptation and learning at the molecular scale. My research presented here is a humble step toward those goals. I will present the design of novel molecular systems made from DNA that exhibit complex neural computation and learning behaviors.
\r\n\r\nChapter 2 covers my contribution to scaling up the computing power of DNA circuits. From bacteria following simple chemical gradients to the brain distinguishing complex odor information, the ability to recognize molecular patterns is essential for biological organisms. This type of information-processing function has been implemented using DNA-based neural networks. Winner-take-all computation has been suggested as a potential strategy for enhancing the capability of DNA-based neural networks. Compared to the linear-threshold circuits and Hopfield networks used previously, winner-take-all circuits are computationally more powerful, allow simpler molecular implementation, and are not constrained by coupling the number of patterns and their complexity, so both a large number of simple patterns and a small number of complex patterns can be recognized. Here, we report a systematic implementation of winner-take-all neural networks based on DNA-strand-displacement reactions. We use a previously developed seesaw DNA gate motif, extended to include a simple and robust component that facilitates the cooperative hybridization involved in selecting a \u2018winner.' We show that with this extended seesaw motif, DNA-based neural networks can classify patterns into up to nine categories. Each of these patterns consists of 20 distinct DNA molecules chosen from the set of 100 that represents the 100 bits in 10x10 patterns, with the 20 DNA molecules selected tracing one of the handwritten digits \u20181\u2019 to \u20189.' The network successfully classified test patterns with up to 30 of the 100 bits flipped relative to the digit patterns \u2018remembered\u2019 during training, suggesting that molecular circuits can robustly accomplish the sophisticated task of classifying highly complex and noisy information on the basis of similarity to a memory.
\r\n\r\nChapter 3 investigates the development of a computational neural network model inspired by biological learning mechanisms, particularly focusing on the new mechanisms for learning in a WTA neural network. The study incorporates novel molecular motifs used in inhibited activators and inhibited weights, designed specifically for training from environmental input patterns. These motifs emulate biological systems by facilitating memory storage and retrieval within DNA-based neural networks, similar to synaptic connections and signal processing observed in living organisms. We assess the function of the individual molecular motifs and characterize their specificity in up to 18-species cross-talk experiments. Furthermore, we characterize the network's performance across a wide array of training and test patterns, mirroring the adaptive responses and diverse conditions encountered by biological systems. Additionally, we analyze the computational efficiency and speed of the learning system, comparing it with both the previous non-learning DNA-based WTA model and a direct weight activation model. By exploring the principles of molecular learning, particularly within winner-take-all neural networks, this study aims to advance computational systems by emulating adaptability and resilience observed in biological organisms using robust, new molecular motifs.
", "doi": "10.7907/529f-kf62", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16438", "collection": "thesis", "collection_id": "16438", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292024-221741183", "primary_object_url": { "basename": "Thesis_Rev_TaraChari.pdf", "content": "final", "filesize": 11821855, "license": "cc_by_nc_nd", "mime_type": "application/pdf", "url": "/16438/1/Thesis_Rev_TaraChari.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Perturbing the Genome: From Bench to Biophysics", "author": [ { "family_name": "Chari", "given_name": "Tara Varada", "orcid": "0000-0002-6953-4313", "clpid": "Chari-Tara-Varada" } ], "thesis_advisor": [ { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "thesis_committee": [ { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "In single-cell genomics, we can simultaneously assay hundreds of thousands of cells, their molecular contents, and how they respond to perturbation, from genetic knockouts to environmental changes. This thesis focuses on how to merge experimental and computational techniques to generate and analyze large-scale perturbation data for high-resolution systems biology. Beginning at the bench, we demonstrate how combining large-scale cell atlas surveys with multi-condition experimentation can illuminate the diversity of cell types across whole organisms and cellular strategies in response to environmental changes and perturbations. We then investigate the limitations of current practice in exploratory analysis, and strategies for determining preservation or distortion of biological insight by these data transformation and dimensionality reduction techniques. To address these limitations, we demonstrate how stochastic biophysical models can rewrite the way we interpret complex perturbation data, taking greater advantage of the diverse molecular measurements to develop biological hypotheses about DNA and RNA regulation in cellular function, development, and disease.
", "doi": "10.7907/5drv-ma07", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16100", "collection": "thesis", "collection_id": "16100", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06092023-193714022", "primary_object_url": { "basename": "schulte_samuel_2023.pdf", "content": "final", "filesize": 154325815, "license": "other", "mime_type": "application/pdf", "url": "/16100/4/schulte_samuel_2023.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "New HCR Technologies: 10-Plex Quantitative Spectral Imaging of RNAs and Proteins; Multiplexed Quantitative Imaging of Protein:Protein Complexes; and Sensitive, Instrument-Free, At-Home Pathogen Detection", "author": [ { "family_name": "Schulte", "given_name": "Samuel Jordan", "orcid": "0000-0001-9982-6504", "clpid": "Schulte-Samuel-Jordan" } ], "thesis_advisor": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Signal amplification based on the mechanism of hybridization chain reaction (HCR) enables researchers to quantitatively image RNA and protein expression in highly autofluorescent biological samples. This thesis extends the capabilities of HCR to three new domains: spectral HCR imaging for quantitative 10-plex immunofluorescence and in situ hybridization in highly autofluorescent samples; imaging of protein:protein complexes using cooperative probes for logical control over HCR signal amplification; and HCR lateral flow tests for sensitive, instrument-free, at-home testing for infectious diseases.
\r\n\r\nWhile 4- or 5-plex imaging is readily achieved using orthogonal HCR systems labeled with spectrally distinct fluorophores, higher levels of multiplexing are challenging due to overlap in the broad excitation and emission spectra of commonly used fluorophores. In Chapter 2, we simultaneously image a combination of 10 protein and RNA targets via spectral imaging with linear unmixing. A combination of 10 reference spectra for 10 fluorophores chosen for optimal unmixing, 10 orthogonal HCR systems, and 11 optimized excitation and emission settings enable robust, user-friendly performance, which is demonstrated in whole-mount zebrafish embryos and mouse brain sections. We validate that unmixed subcellular voxel intensities enable accurate and precise relative target quantitation with subcellular resolution across all 10 channels and demonstrate single-molecule sensitivity and resolution for absolute RNA quantitation.
\r\n\r\nIn Chapter 3, we introduce an enzyme-free method for multiplexed imaging of protein:protein complexes using split-initiator HCR signal amplification. Antibodies specific to each protein of the complex carry fractional initiators that become colocalized upon introduction of a DNA ruler strand to form a full HCR initiator and trigger growth of a tethered amplification polymer. Automatic background suppression is present throughout the protocol, as split-initiator antibody probes that bind to the sample nonspecifically or to isolated protein targets are too far apart to become colocalized by the ruler strand, precluding colocalization of a full initiator and preventing HCR signal amplification. We demonstrate the technique with high signal-to-background in adherent mammalian cells, pro-T cells, and highly autofluorescent formalin-fixed paraffin-embedded human breast tissue sections. Leveraging existing orthogonal HCR amplifiers, we design three orthogonal cooperative junctions for simultaneous 3-plex detection of protein:protein complexes. We validate that quantitative subcellular voxel intensities are generated, allowing for built-in relative quantitation of protein:protein complexes within the spatial context of the sample. Lastly, we demonstrate simultaneous detection of protein targets, RNA targets, and protein:protein complexes via a unified protocol for HCR immunofluorescence, in situ hybridization, and protein:protein complex imaging.
\r\n\r\nIn Chapter 4, we enhance the sensitivity of conventional unamplified lateral flow tests for at-home infectious disease testing by developing an amplified assay with isothermal, enzyme-free signal amplification based on the mechanism of HCR. Traditional lateral flow tests are amenable to at-home testing and return a result within 10\u201315 minutes but demonstrate a high false-negative rate (e.g., 25-50% for SARS-CoV-2) due to the absence of signal amplification. The HCR lateral flow assay we develop maintains the simplicity of the conventional lateral flow assay user experience via a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 minutes. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/\u03bcL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/\u03bcL, 1000 copies/\u03bcL, 2000 copies/\u03bcL, 2000 copies/\u03bcL, and 20,000 copies/\u03bcL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious diseases.
", "doi": "10.7907/nzk8-2d38", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15093", "collection": "thesis", "collection_id": "15093", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01272023-184413283", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 16608292, "license": "other", "mime_type": "application/pdf", "url": "/15093/1/Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Towards Integrated Molecular Machines: Structural, Mechanical, and Computational Motifs", "author": [ { "family_name": "Sarraf", "given_name": "Namita", "orcid": "0000-0001-8692-7429", "clpid": "Sarraf-Namita" } ], "thesis_advisor": [ { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The programmability of DNA has made it well-suited for building molecular machines, performing nanoscale self-assembly, and computing via biochemical circuits. In the last few decades, great strides have been made in characterizing the interactions between DNA molecules such that they can be predicted and engineered.
\r\n\r\nThe development of frameworks for those interactions has enabled the construction of more complex molecular systems that can execute specified programs. Such programs have included mechanical tasks, like walking and sorting cargo; assembly and reconfiguration of 2D and 3D shapes; and computation, like Boolean logic and pattern recognition.
\r\n\r\nHowever, the continuing development of more complex molecular programs relies upon expanding the modules available for molecular systems to use to execute them. Expanded functionality of mechanical, structural, and computation modules are required in order to build compound systems that can interact with the physical world, reconfigure, and analyze signals in a variety of interesting ways. In this dissertation, we will discuss our contributions to this effort, which include exploring a motif for molecular robotic behavior, characterizing tile-tile interactions, and developing new capabilities for bimolecular circuits.
\r\n\r\nWithin the framework of a maze-solving molecular robot, we aim to implement walking behavior on DNA origami that introduces a surface modification via a four-way strand displacement reaction. Surprisingly, our experiments suggest that the walking behavior is at least two orders of magnitude slower than expected. To understand why, we quantitatively explore to what extent the speed and completion level of the robot can be modulated by design considerations such as toehold lengths, track redundancy, and strand purity. Another factor affecting the reaction rate is the number of tethering points, and we demonstrate an order of magnitude speed up in the four-way strand displacement reaction when we remove one tethering point. The characterization of a surface-modifying four-way strand displacement reaction is a useful tool for the continued development of molecular robots with more complex functionality.
\r\n\r\nFree-floating DNA origami tiles, called invaders here, can swap out DNA origami tiles within larger assemblies via a technique called tile displacement, which has previously been demonstrated using single tile and dimer invaders with 4- and 9-tile arrays. We introduce initial structures and invading assemblies with more complex shapes. We explore the robustness of this reaction by testing a variety of edge configurations and comparing their reaction rates. We demonstrate tunable growth of one of the invaders, which can grow into polymers of arbitrary length or close into 3D structures. By a tile displacement reaction, we reconfigure the 3D structures into 2D. The invaders with complex shapes are able to reconfigure the original tile assembly at rates comparable to simpler tile displacement reactions, and two reconfiguration events can take place sequentially or simultaneously.
\r\n\r\nFinally, we build two new modules for use with biochemical circuits. The first, a loser-take-all circuit, yields binary outputs indicating which analog signal is the smallest among all inputs. We implement a signal reversal function that converts the smallest input to the largest output, which can then be composed with a previously developed winner-take-all function to achieve loser-take-all. By making concentration adjustments, we can mitigate biases in the circuit that are a result of sequence-dependent different in reaction rates. We experimentally demonstrate a three-input loser-take-all circuit with nine input combinations. With further development, this circuit could be used to implement the activation function in neural networks that perform pattern classification according to which memory an input pattern is least similar to.
\r\n\r\nThe second circuit processes information using temporary memory. We design and implement a circuit that outputs distinct logic decisions based on relative timing information of a pair inputs and their logic values. We show that we can mitigate crosstalk in the circuit by utilizing mismatches and adjusting toehold lengths. The circuit is able to display clear ON-OFF separation at time intervals as short as one minute between the two inputs arriving.
", "doi": "10.7907/cdwp-c709", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:13838", "collection": "thesis", "collection_id": "13838", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07082020-113341068", "type": "thesis", "title": "Guiding Self-Organization in Active Matter with Spatiotemporal Boundary Conditions", "author": [ { "family_name": "Ross", "given_name": "Tyler David", "orcid": "0000-0002-7872-3992", "clpid": "Ross-Tyler-David" } ], "thesis_advisor": [ { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "In this thesis, I demonstrate that self-organized structures and forces can be guided by modulating the interactions between force-generating molecules in space and time. The physics of self-organizing systems is an open frontier. We do not have a complete set of principles that can describe how a dynamic structure forms based on the non-equilibrium dynamics of its constituent components. Yet, living systems appear to depend on some set of rules of self-organization in order to reliably carry out their mechanical functions. Force-generating, active, molecules in the form of motor proteins and filamentous polymers are responsible for performing fundamental tasks in living matter, such as locomotion and division. While it is known that the regulation of motor-filament interactions is necessary to achieve the dynamic structures that drive movement and propagation, the role of spatial and temporal patterning in self-organizing systems has not been explored. I design a artificial system of purified molecules where the interactions between motors and filaments are toggled with light. By patterning molecular interactions in space and time, I show that it is possible to localize the formation of spherically symmetric asters, which can be moved, merged, and used to generate advective fluid flows. The ability to pattern molecular interactions in space and time offers a new perspective in the search for principles of active self-organization. Spatial and temporal control makes it possible to start distilling how the interactions between active molecules determine the mesoscopic behaviors of self-organized structures. These rules ultimately govern the physics of living matter and may eventually be harnessed to build new materials and cell-like machines.
", "doi": "10.7907/q85h-j730", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14232", "collection": "thesis", "collection_id": "14232", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022021-043318684", "type": "thesis", "title": "Enhanced Noninvasive Imaging of Acoustic Biomolecules", "author": [ { "family_name": "Sawyer", "given_name": "Daniel Patrick", "orcid": "0000-0003-2926-191X", "clpid": "Sawyer-Daniel-Patrick" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The extensive scientific interest in cellular and biomolecular processes is due in large part to the importance of such processes deep inside living organisms, in the context of both health and disease. However, most methods for imaging cellular processes such as gene expression have relied on fluorescent proteins and other optical reporters that, while providing a direct optical readout of the biomolecular environment in cells readily exposed to light, have greatly limited performance in large animals due to the poor penetration of visible light beyond 1 mm of biological tissue. In contrast, ultrasound is widely used to noninvasively image tissue deep inside living organisms but has rarely been used to investigate cellular function due a lack of acoustic reporters whose production and properties are coupled to biomolecular events. Recently, the first acoustic reporter genes (ARGs) were developed for ultrasound imaging of a unique class of air-filled protein nanostructures known as gas vesicles, or GVs, which scatter sound waves when expressed in bacterial and mammalian cells. ARGs allow gene expression to be visualized with ultrasound similar to how green fluorescent protein (GFP) allowed gene expression to be visualized with light. However, ARGs will have limited utility in practical applications involving living organisms without ultrasound imaging methods providing the specificity to reliably distinguish GVs from surrounding tissue and the sensitivity to detect GVs at low concentrations.
\r\n\r\nIn this thesis, we present two novel ultrasound imaging methods that exploit the unique nonlinear physical properties of gas vesicles to enhance image quality in situations that pose challenges for conventional imaging methods. In Chapter 1, we address the problem of distinguishing GVs from tissue with cross-Amplitude Modulation (xAM), an ultrasound pulse sequence that uses X-waves to isolate the signal generated by reversible buckling of the GV shell while cancelling scattering and artifacts from tissue. In Chapter 2, we present an application of xAM to imaging of dynamic biomolecular processes. We show that, when GVs are engineered such that buckling is induced by enzyme activity, xAM can visualize enzymatic processes deep inside living animals. In Chapter 3, we address the problem of detecting very low concentrations of ARG-expressing cells with Burst Ultrasound Reconstructed with Signal Templates (BURST), an imaging method that exploits the strong, transient signals generated during sudden GV collapse under acoustic pressure by unmixing the temporal dynamics of such signals from background scattering. BURST imaging improves cellular sensitivity by more than 1000-fold and, in dilute cell suspensions, enables the detection of gene expression in individual bacteria and mammalian cells. In Chapter 4, we present an application of an early formulation of BURST to imaging gene expression in mammalian cells. We use this imaging method to visualize vascularization patterns in tumors containing mammalian cells expressing acoustic reporter genes.
", "doi": "10.7907/p52e-qv56", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13687", "collection": "thesis", "collection_id": "13687", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04292020-052418975", "type": "thesis", "title": "Formal Design and Analysis for DNA Implementations of Chemical Reaction Networks", "author": [ { "family_name": "Johnson", "given_name": "Robert Francis", "orcid": "0000-0002-5340-8347", "clpid": "Johnson-Robert-Francis" } ], "thesis_advisor": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Bruck", "given_name": "Jehoshua", "orcid": "0000-0001-8474-0812", "clpid": "Bruck-J" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "In molecular programming, the Chemical Reaction Network model is often used to describe systems of interacting molecules. This model can describe either real systems, allowing us to analyze and determine their computational function; or describe hypothetical systems, with known computational function but perhaps no known physical example. One significant breakthrough in the field is that any Chemical Reaction Network can be approximated by a system using DNA Strand Displacement mechanisms. This allows the Chemical Reaction Network model to be treated like a programming language, where programs can be written in the abstract and then compiled into physical molecules. Given a programming language and a proof-of-concept compiler, one would want to take the compiler from the proof-of-concept stage into a more reliable, more systematic, and better understood process. This thesis is made up of my contributions to that effort.
\r\n\r\nFirst, given a programming language and a compiler, it would be useful to formally verify that the compiler is correct. My collaborators, Qing Dong and Erik Winfree, and I defined a Chemical Reaction Network-specific form of bisimulation equivalence, which can compare two such networks and verify that one is (or is not) a correct implementation of the other. For example, the compiler-produced DNA circuit can be verified as an implementation of its abstract program, although this is not the only possible use. After defining this concept of equivalence, we show that it can be checked by algorithm; although various parts of the problem are NP-complete or PSPACE-complete, we give algorithms that meet these lower bounds. We also prove a number of interesting properties of Chemical Reaction Network bisimulation equivalence, including transitivity and modularity properties which are particularly useful for stepwise checking of large systems. We further extend this bisimulation method to linear Polymer Reaction Networks, a strictly more powerful abstraction which has been occasionally used in molecular programming. Again we prove complexity hardness results, which in this case are as expected uncomputable in the general case; however, many practical systems can still be verified, and we give one such example. Finally, we use bisimulation to identify a class of single-locus networks that are practical to implement. Thus we show a method of verification which can simplify use of the above-mentioned compiler by proving general statements of correctness about its results.
\r\n\r\nSecond, given a programming language and a concept of compiling it, it would be useful to optimize the result of the compilation. One particular area of optimization is the number of DNA strands per prepared complex; some experiments suggest that systems with no more than 2 strands per complex are more robust. Lulu Qian and I developed some proposed DNA Strand Displacement schemes for general Chemical Reaction Network implementations with no more than 2 strands per complex, and a number of other desirable properties. Meanwhile, having been shown to be useful for many reasons, the mechanisms of DNA Strand Displacement have recently been formalized, abstracted, and analyzed. I show that this formalization, combined with the bisimulation methods above, can prove various statements about the limits of DNA Strand Displacement systems. For example, a set of desirable conditions including the 2-strand limit cannot be achieved by any general Chemical Reaction Network implementation scheme. I also observe that two of the new schemes we discovered, each meeting all but one condition of the impossible set, were found in the process of coming up with this proof. I thus argue that through formalization of DNA Strand Displacement we can have a more systematic method of finding and designing molecular programs, and of knowing when the programs we want do not exist.
", "doi": "10.7907/a74v-kv80", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:10980", "collection": "thesis", "collection_id": "10980", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312018-112853523", "primary_object_url": { "basename": "Philip Petersen Thesis Final 2.pdf", "content": "final", "filesize": 12657109, "license": "other", "mime_type": "application/pdf", "url": "/10980/1/Philip Petersen Thesis Final 2.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Engineering Molecular Self-assembly and Reconfiguration in DNA Nanostructures", "author": [ { "family_name": "Petersen", "given_name": "Philip Fai", "orcid": "0000-0002-9912-389X", "clpid": "Petersen-Philip-Fai" } ], "thesis_advisor": [ { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Smart electronics have developed ubiquitously to assist people in everything from navigation to health monitoring. The rise of complex electronics relied on rational design of platforms to build ever larger and more complex circuit networks and for frameworks to test those electronics. Biochemical circuits have also seen dramatic advancement in the last two decades within the field of DNA nanotechnology. As with electronics, DNA nanotechnology applied rational design to DNA molecules to build ever more complex biochemical networks that, beyond current electronics, also retain a significant measure of biological compatibility and plasticity akin to many networks of biological origin. Well situated for promising applications in diagnostics and therapeutics, advancing DNA nanotechnology devices will also rely upon larger platforms and testing frameworks.
\r\n\r\nIn roughly the last decade, researchers have been building upon the invention of DNA origami, a technique allowing the robust construction of biomolecular nano-structures capable of precise nanometer positioning of proteins, nanoparticles, and other molecules. DNA circuits have computed on the nanostructures; DNA robots have moved nanoparticles, made choices, and have even sorted cargo on the surface of a nanostructure. The complexity of circuits and devices continues to rise.
\r\n\r\nIn this thesis, we will discuss our contributions to the field of DNA nanotechnology by developing design rules and systematic approaches to controlling nanostructure complex assembly. These rules and approaches allow for the construction of molecular structures with a tunable diversity, large systems approaching the size of bacteria yet retaining nanometer precision, and biological plasticity inspired dynamic systems for arbitrary reconfiguration.
\r\n\r\nUsing a DNA origami tile tailored for array formation with a high continuous surface area, we create a framework inspired from molecular stochasticity for programming DNA array formation and gaining control over diversity of global properties through simple local rules. Three general forms of planar networks, random loops, mazes, and trees, were manipulated on the micron scale upon the self-assembled DNA arrays. We demonstrate control of several properties of the networks, such as branching rules, growth directions, the proximity between adjacent networks, and size distributions. The large diversity, in principle, allows for a wide, but tunable, testing environment for molecular circuits. By further applying these principles to subunits of finite assemblies, variable components may be mixed with fixed components potentially opening additional applications in high throughput device or drug screening.
\r\n\r\nNext we turned to expanding the platform size biochemical circuits may be built upon. While DNA origami allows nanometer precise placement, the size remains roughly below 0.05 um2. Toward making large arbitrarily complex structures with only a set of simple tiles, multi-stage self-assembly has been explored in theory and for small DNA tiles. None were successful experimentally with DNA origami. We developed a strategy for DNA origami: a simple rule set applied recursively in each stage of a hierarchical self-assembly process, and to significantly reduce costs, a constant set of unique DNA strands regardless of size. We also developed a software tool to automatically compile a designed surface pattern into experimental protocols. We experimentally demonstrated DNA origami arrays approaching the size of small bacteria, 0.5 um2, with several arbitrary patterns, each consisting of 8,704 specifically chosen pixel locations with nanometer precision, including a bacteria sized portrait of a bacteria. The large platform opens the door to more advanced molecular circuits for applications such as diagnostics.
\r\n\r\nFinally we demonstrated control over the dynamics of DNA origami reconfiguration in tile arrays. In an approach we call DNA tile displacement, we showed that a DNA origami array may have tiles arbitrarily replaced by another tile, including tiles of another shape or surface pattern. We also demonstrated control over the kinetics of tile displacement and performed several general purpose reconfigurations of DNA nanostructures. Examples include sequential reconfiguration, competitive reconfiguration, cooperative reconfiguration, and finally the scalability of multi-step reconfiguration as demonstrated through a fully playable nano-scale biomolecular tic-tac-toe game. The major ramifications are a plasticity more common to biology than to electronics\u2014molecular platforms with arbitrary patterning that can reconfigure an arbitrary part of the nanostructure in an arbitrary order based on environmental signals. In principle, such reconfiguration can allow advanced circuits with the capacity to adapt to environmental needs or heal damaged components.
\r\n", "doi": "10.7907/7FXP-8402", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10323", "collection": "thesis", "collection_id": "10323", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082017-194534497", "primary_object_url": { "basename": "Anu_Thubagere_BBE.pdf", "content": "final", "filesize": 18346331, "license": "other", "mime_type": "application/pdf", "url": "/10323/1/Anu_Thubagere_BBE.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Programming Complex Behavior in DNA-based Molecular Circuits and Robots", "author": [ { "family_name": "Thubagere Jagadeesh", "given_name": "Anupama", "clpid": "Thubagere-Jagadeesh-Anu" } ], "thesis_advisor": [ { "family_name": "Qian", "given_name": "Lulu", "clpid": "Qian-Lulu" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "clpid": "Rothemund-P-W-K" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Qian", "given_name": "Lulu", "clpid": "Qian-Lulu" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Integrated electronic circuits, like those found in cellphones and computers, are ubiquitous in our information-driven society. The success of electronics has, in part, been due its modular architecture that enables individual components to be independently improved while the overall device functionality remains unchanged. Over the last two decades the emerging field of dynamic DNA nanotechnology has been trying to apply the underlying philosophy of electronics to biochemical circuits. DNA nanotechnology employs rationally designed DNA molecules as building blocks of biochemical circuits that can, in principle, enable powerful applications like diagnostics and therapeutics.
\r\n\r\nResearchers in the field of DNA nanotechnology have developed simple elements to construct biomolecular systems with desired functions. They have also developed molecular compilers for defining design principles. The cost of DNA synthesis has decreased by over three orders of magnitude in the past decade. This has lead to a non-trivial number of small scale circuits, like DNA-based logic gates and chemical oscillators, being implemented. However, the scalability of this approach has yet to be clearly demonstrated. n this thesis, we will discuss our main contributions to facilitating the advancement of DNA nanotechnology by developing systematic approaches for constructing modular DNA building blocks. These modules can be used to construct biochemical circuits and molecular robotic systems. The performance of the modules can be individually tuned and integrated into large-scale systems.
\r\n\r\nUsing automated circuit-design software and cheap unpurified DNA, we demonstrated the design and construction of a complex synthetic biochemical circuit consisting of 78 distinct DNA species. The circuit is capable of computing the transition rules of a cell updating its state based on its neighboring cells, defined in a classic computational model called cellular automata. Using a bottom-up approach, we first characterized the component necessary for basic Boolean logic computation. We then systematically integrated more circuit elements and eventually constructed the full circuit. By developing a systematic procedure for building DNA-based circuits using unpurified components, we significantly simplified the experimental procedure. By using unpurified DNA components, we reduced the cost and technical barrier for circuit construction, thus making the design and synthesis of complex DNA circuits accessible to even novice researchers.
\r\n\r\nNext we demonstrated a cargo sorting DNA nano-robot, using a simple algorithm and modular building blocks. The DNA robot has a leg and two foot domains for exploring a two-dimensional DNA origami surface, and an arm and hand domain for picking up randomly located cargos and dropping them off at their designated locations. It is completely autonomous and is programmed to perform a random walk without requiring an external energy source. Further, we demonstrated sorting multiple copies of two distinct cargo species on the same origami. Additionally, by compartmentalizing each sorting task on a single origami, we showed that two distinct sorting tasks can be implemented on different origami simultaneously in the same test tube. The recognition of a cargo is embedded in its destination, therefore it is possible to scale up the system simply by having multiple types of cargos. The same robot design can be used for performing multiple instances of distinct tasks in parallel. The different modules can be integrated to perform diverse functions, including applications in time-release targeted therapeutics.
", "doi": "10.7907/Z9WD3XMS", "publication_date": "2017-06-16", "thesis_type": "phd", "thesis_year": "2017" } ]