[ { "id": "https://thesis.library.caltech.edu/id/eprint/15086", "eprint_id": 15086, "rev_number": 64, "documents": [ { "id": "/id/document/138072", "doc_id": 138072, "rev_number": 4, "files": [ { "id": "/id/file/402927", "fileid": 402927, "datasetid": "document", "objectid": 138072, "filename": "Thesis-Submit.zip", "mime_type": "application/zip", "hash": "281b699e9507c48a6cb6c9984503d4de", "hash_type": "MD5", "filesize": 512270810, "mtime": "2023-01-17 20:14:19", "url": "/15086/2/Thesis-Submit.zip" } ], "eprint_id": 15086, "pos": 2, "placement": 2, "mime_type": "application/zip", "format": "application/zip", "format_desc": "Thesis Source", "language": "en", "security": "internal", "license": "other", "main": "Thesis-Submit.zip", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "content": "archival" }, { "id": "/id/document/138076", "doc_id": 138076, "rev_number": 4, "files": [ { "id": "/id/file/402991", "fileid": 402991, "datasetid": "document", "objectid": 138076, "filename": "JH_Thesis_v4.5.pdf", "mime_type": "application/pdf", "hash": "eacd4ff9f52cb1fec0e515397b80313b", "hash_type": "MD5", "filesize": 169170514, "mtime": "2023-01-18 21:31:19", "url": "/15086/3/JH_Thesis_v4.5.pdf" } ], "eprint_id": 15086, "pos": 3, "placement": 3, "mime_type": "application/pdf", "format": "application/pdf", "language": "en", "security": "public", "license": "other", "main": "JH_Thesis_v4.5.pdf", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "content": "final" }, { "id": "/id/document/138077", "doc_id": 138077, "rev_number": 1, "files": [ { "id": "/id/file/402994", "fileid": 402994, "datasetid": "document", "objectid": 138077, "filename": "indexcodes.txt", "mime_type": "text/plain", "hash": "9ced9f30cb48bd01c563e61e68fecb70", "hash_type": "MD5", "filesize": 42214, "mtime": "2023-01-18 21:41:30", "url": "/15086/4/indexcodes.txt" } ], "eprint_id": 15086, "pos": 4, "placement": 4, "mime_type": "text/plain", "format": "other", "format_desc": "Generate index codes conversion from application/pdf to indexcodes", "language": "en", "security": "public", "license": "other", "main": "indexcodes.txt", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/138076" }, { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/138076" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/138076" } ] } } ], "eprint_status": "archive", "userid": 10671, "dir": "disk0/00/01/50/86", "datestamp": "2023-01-20 18:16:12", "lastmod": "2023-11-08 00:43:31", "status_changed": "2023-01-20 18:16:12", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "jiningh@gmail.com", "id": "Huang-Jining", "name": { "family": "Huang", "given": "Jining" }, "orcid": "0000-0002-3798-4790", "show_email": "YES" } ] }, "title": "Applications of Dynamic Nucleic Acid Nanotechnology in Closed-Loop Genetic Circuits and Detection of Viral Pathogens", "ispublished": "unpub", "full_text_status": "public", "keywords": "DNA; RNA; dynamic RNA nanotechnology; CRISPR; genetic feedback circuits; synthetic genetic circuits; synthetic biology; small conditional RNAs; lateral flow assay; hybridization chain reaction (HCR); rapid antigen test; SARS-CoV-2 RNA genome; secondary structure; base-pairing; hybridization; complex ensemble; test tube ensemble; multi-tube ensemble; equilibrium; concentration; ensemble defect; analysis; design; reaction pathway engineering", "abstract": "
Nucleic acid nanotechnologies have provided a platform where biologically relevant molecules can be engineered to perform programmable functions. Relative to proteins, complex nucleic acid-based systems can be designed more readily due to the countable nature of base-pairing interactions and readily available physical models. These features of nucleic acids enable us to design novel interaction pathways and functions by providing well-behaved molecular mechanisms. Two examples of these mechanisms are the conditional guide RNA (cgRNA) and the hybridization chain reaction (HCR). A cgRNA is a conditional programmable regulator where an expressed RNA trigger can conditionally turn on or off transcriptional regulation. HCR is a molecular mechanism for in vitro and in situ amplification of signals to spatially identify proteins, RNA, or DNA in a sample. This thesis will first demonstrate the use of these nucleic acid molecular mechanisms in closed-loop genetic circuits and infectious disease testing using cgRNAs and HCR, respectively, then provide updated tools for the nucleic acid design community to exploit the programmable nature of nucleic acids.
\r\n\r\nWe begin by demonstrating the use of conditional programmable cgRNAs in closed-loop genetic circuits. Synthetic genetic circuits allow scientists to engineer arbitrary molecular interactions in living organisms. Feedback circuits in particular are recurrently found in nature and enable useful functionalities. However, protein components of genetic circuits cannot be designed scalably, are often mined from preexisting genomes, and present difficulties in being biologically orthogonal to themselves or the host organism. We are motivated to address these limitations by using orthogonal nucleic acid circuits created de novo. One potential component of these circuits are conditional guide RNAs (cgRNAs). cgRNAs are switchable transcriptional regulators, and this allows gene expression to be modulated through the expression of a small RNA trigger. Here we assess cgRNAs as a component for feedback genetic circuits. As an initial demonstration of cgRNA synthetic circuits, we built and validated a simple threshold circuit and demonstrated its orthogonality and scalability by showing independent circuit functions of two switches in a single cell. We also created a larger toggle switch that is made from the same components as the previous switches. These experiments show the orthogonality and feedback capabilities of cgRNAs will position them as a composable component for scalable synthetic biology.
\r\n\r\nWe then used the hybridization chain reaction mechanism to develop an adaptable and sensitive test for the detection of SARS-CoV-2. The lateral flow assay format enables rapid, instrument-free, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience after the test begins is maintained by using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. Prior to starting the test, a 15-minute heat step is required. Detecting gamma-irradiated SARS-CoV-2 virions in an extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/\u00b5L using nucleic acid probes to target the SARS-CoV-2 RNA genome. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies to target the viral nucleocapsid protein exhibit limits of detection of 500 copies/\u00b5L, 1000 copies/\u00b5L, 2000 copies/\u00b5L, 2000 copies/\u00b5L, and 20,000 copies/\u00b5L. By swapping out nucleic acid probes to target different pathogens, amplified HCR lateral flow assays offer a platform for adaptable and sensitive at-home testing for emergent diseases.
\r\n\r\nComponents for the previous two projects are designed and analyzed with NUPACK. NUPACK is a growing software suite for the analysis and design of nucleic acid structures, devices, and systems serving the needs of researchers in the fields of nucleic acid nanotechnology, molecular programming, synthetic biology, and across the life sciences. NUPACK algorithms are unique in treating complex and test tube ensembles containing arbitrary numbers of interacting strand species, providing crucial tools for capturing concentration effects essential to analyzing and designing the intermolecular interactions that are a hallmark of these fields. The all-new NUPACK web app (nupack.org) has been re-architected for the cloud, leveraging a cluster that scales dynamically in response to user demand to enable rapid job submission and result inspection even at times of peak user demand. The web app exploits the all-new NUPACK 4 scientific code base as its backend, offering enhanced physical models (coaxial and dangle stacking sub-ensembles), dramatic speedups (20-120\u00d7 for test tube analysis), and increased scalability for large complexes.
", "date": "2023", "date_type": "degree", "id_number": "CaltechTHESIS:01172023-195222304", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:01172023-195222304", "related_url": { "items": [ { "description": "Article adapted for Chapter 3.", "type": "doi", "url": "https://doi.org/10.1101/2022.09.18.508442" }, { "description": "Article adapted for Chapter 4.", "type": "doi", "url": "https://doi.org/10.26434/chemrxiv-2022-xv98l" } ] }, "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "Defense Advanced Research Projects Agency (DARPA)", "grant_number": "HR0011-17-2-0008" }, { "agency": "Center for Environmental Microbiology Interactions" }, { "agency": "Beckman Institute (Programmable Molecular Technology Center)" }, { "agency": "Shurl and Kay Curci Foundation" }, { "agency": "Richard N. Merkin Institute for Translational Research" }, { "agency": "National Aeronautics and Space Administration", "grant_number": "NNX16AO69A" }, { "agency": "National Institutes of Health (NIH)", "grant_number": "R01 EB006192" }, { "agency": "National Science Foundation", "grant_number": "NSF-OAC-1835414" }, { "agency": "National Science Foundation", "grant_number": "NSF-CHE-1643606" }, { "agency": "National Science Foundation", "grant_number": "NSF-CCF-1317694" }, { "agency": "National Science Foundation", "grant_number": "NSF-ACI-1548562" }, { "agency": "National Institutes of Health (NIH)", "grant_number": "T32 GM007616" }, { "agency": "Amazon Web Services" } ] }, "collection": "CaltechTHESIS", "reviewer": "Melissa Ray", "suggestions": "Released 2023-02-02 after 1-week embargo.", "deposited_by": "Jining Huang", "deposited_on": "2023-01-20 18:16:12", "doi": "10.7907/54tw-ym95", "divisions": { "items": [ "div_bbe" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "winfree@caltech.edu", "id": "Winfree-E", "name": { "family": "Winfree", "given": "Erik" }, "orcid": "0000-0002-5899-7523", "role": "chair" }, { "email": "murray@caltech.edu", "id": "Murray-R-M", "name": { "family": "Murray", "given": "Richard M." }, "orcid": "0000-0002-5785-7481", "role": "member" }, { "email": "melowitz@caltech.edu", "id": "Elowitz-M-B", "name": { "family": "Elowitz", "given": "Michael B." }, "orcid": "0000-0002-1221-0967", "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2022-12-02", "gradofc_approval_date": "2023-01-20", "review_status": "approved", "option_major": { "items": [ "bioeng" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2023", "author_list": "Huang, Jining", "advisor_list": "Pierce, Niles A.", "comittee_list": "Winfree, Erik; Murray, Richard M.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/15070", "eprint_id": 15070, "rev_number": 50, "documents": [ { "id": "/id/document/137996", "doc_id": 137996, "rev_number": 4, "files": [ { "id": "/id/file/402307", "fileid": 402307, "datasetid": "document", "objectid": 137996, "filename": "Thesis.zip", "mime_type": "application/zip", "hash": "afbb851aaebd070a09ea6ec437358933", "hash_type": "MD5", "filesize": 5815341, "mtime": "2022-12-02 01:10:55", "url": "/15070/2/Thesis.zip" } ], "eprint_id": 15070, "pos": 2, "placement": 2, "mime_type": "application/zip", "format": "application/zip", "language": "en", "security": "internal", "license": "other", "main": "Thesis.zip", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "content": "archival" }, { "id": "/id/document/138195", "doc_id": 138195, "rev_number": 6, "files": [ { "id": "/id/file/403899", "fileid": 403899, "datasetid": "document", "objectid": 138195, "filename": "Thesis_20230228ZC.pdf", "mime_type": "application/pdf", "hash": "5f2e55e3ff70056e658b7aa59a2acd91", "hash_type": "MD5", "filesize": 9080457, "mtime": "2023-03-01 00:26:43", "url": "/15070/3/Thesis_20230228ZC.pdf" } ], "eprint_id": 15070, "pos": 3, "placement": 3, "mime_type": "application/pdf", "format": "application/pdf", "language": "en", "security": "public", "license": "other", "main": "Thesis_20230228ZC.pdf", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "content": "final" }, { "id": "/id/document/138196", "doc_id": 138196, "rev_number": 1, "files": [ { "id": "/id/file/403904", "fileid": 403904, "datasetid": "document", "objectid": 138196, "filename": "indexcodes.txt", "mime_type": "text/plain", "hash": "778c0758eaa70b930ec8c4c632bffbd7", "hash_type": "MD5", "filesize": 40389, "mtime": "2023-03-01 00:37:00", "url": "/15070/4/indexcodes.txt" } ], "eprint_id": 15070, "pos": 4, "placement": 4, "mime_type": "text/plain", "format": "other", "format_desc": "Generate index codes conversion from application/pdf to indexcodes", "language": "en", "security": "public", "license": "other", "main": "indexcodes.txt", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/138195" }, { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/138195" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/138195" } ] } } ], "eprint_status": "archive", "userid": 11799, "dir": "disk0/00/01/50/70", "datestamp": "2023-03-24 17:31:58", "lastmod": "2023-11-08 00:43:31", "status_changed": "2023-03-24 17:31:58", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "zchen@alumni.caltech.edu", "id": "Chen-Zhewei", "name": { "family": "Chen", "given": "Zhewei" }, "orcid": "0000-0002-7422-095X", "show_email": "YES" } ] }, "title": "Engineering Conditional Guide RNAs for Cell-Selective Regulation of CRISPR/Cas9", "ispublished": "unpub", "full_text_status": "public", "keywords": "nucleic acid nanotechnology\r\nCRISPR Cas9\r\nRNA engineering", "abstract": "CRISPR/Cas9 is a versatile platform for implementing diverse modes of genetic perturbation such as gene silencing, induction, deletion, or replacement. This technology is popularly used in developmental biology to probe genetic circuitry via constitutive gene knockdown. Global gene silencing could introduce artifacts in the study of developmental regulatory pathways, and this motivates the development of cell-selective gene editing. Our lab has recently created conditional guide RNAs (cgRNA) that enable CRISPR/Cas9 systems to silence a desired gene Y conditioned on the detection of an RNA transcript X inside of a cell. cgRNA systems were discovered via insertion and deletion mutations that systematically explored the structure function of the guide RNA. Nucleic acid engineering software (NUPACK) was used to generate orthogonal libraries of cgRNA molecules that executed both ON \u2192 OFF logic (conditional inactivation by an RNA trigger) and OFF \u2192 ON logic (conditional activation by an RNA trigger). A dCas9-based RFP silencing assay in bacteria was developed and used to show these cgRNA sequences were functional and could detect short exogenous trigger sequences in an orthogonal and doseresponsive manner. Subsequent studies on cgRNA structure and function enabled us to engineer next-generation systems that have fewer constraints on the trigger sequence or structure. These next-generation cgRNAs were tested against short synthetic mRNA transcripts, truncated sub-sequences of endogenous mRNAs, and full-length endogenous mRNAs. Synthetic mRNA transcripts were used to study the effect of protein translation on trigger RNA binding. cgRNAs were capable of detecting synthetic sequences embedded in the 3\u2032 UTR of fluorescent protein mRNAs. cgRNAs could also detect short synthetic mRNAs or truncated subsequences from endogenous mRNAs. However, the detection of native full-length endogenous mRNAs remained challenging because we cannot reliably predict the local structure of sub-sequences within a long RNA transcript. High-throughput cgRNAscreening may prove necessary for finding accessible binding sites onmRNA transcripts. Nevertheless, cgRNA functionalities could be useful in developmental biology by enabling precision perturbation of regulatory events, linking guide RNA activity to an RNA marker X correlated to a specific cell type or temporal expression pattern. This work opens the possibility for future applications such as cell-selective gene therapies.
", "date": "2023", "date_type": "degree", "id_number": "CaltechTHESIS:12022022-010002116", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:12022022-010002116", "related_url": { "items": [ { "description": "Article adapted for ch. 2", "type": "doi", "url": "https://doi.org/10.1021/acscentsci.9b00340" } ] }, "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "Defense Advanced Research Projects Agency (DARPA)", "grant_number": "HR0011-17-2-0008; the findings are those of the authors and should not be interpreted as representing the official views or policies of the US Government" }, { "agency": "Rosen Bioengineering Center" }, { "agency": "Molecular Programming Project (NSF)", "grant_number": "NSF-CCF-1317694" }, { "agency": "Resnick Sustainability Institute" }, { "agency": "Biotechnology Leadership Program (National Institute of General Medical Sciences)", "grant_number": "NIH-T32GM112592A" } ] }, "collection": "CaltechTHESIS", "reviewer": "Melissa Ray", "suggestions": "Released 2023-03-31 after 1-week embargo.\r\n\r\n// Permanent non-caltech email is imap.zchen@gmail.com but author wanted to publish the alumni email address for the public.", "deposited_by": "Zhewei Chen", "deposited_on": "2023-03-24 17:31:58", "doi": "10.7907/cajs-d417", "divisions": { "items": [ "div_bbe" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "murray@caltech.edu", "id": "Murray-R-M", "name": { "family": "Murray", "given": "Richard M." }, "orcid": "0000-0002-5785-7481", "role": "chair" }, { "email": "pwkr@dna.caltech.edu", "id": "Rothemund-P-W-K", "name": { "family": "Rothemund", "given": "Paul W. K." }, "orcid": "0000-0002-1653-3202", "role": "member" }, { "email": "haybruce@caltech.edu", "id": "Hay-B-A", "name": { "family": "Hay", "given": "Bruce A." }, "orcid": "0000-0002-5486-0482", "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2022-12-15", "gradofc_approval_date": "2024-03-24", "review_status": "approved", "option_major": { "items": [ "bioeng" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2023", "author_list": "Chen, Zhewei", "advisor_list": "Pierce, Niles A.", "comittee_list": "Murray, Richard M.; Rothemund, Paul W. 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"http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/139824" } ] } } ], "eprint_status": "archive", "userid": 11306, "dir": "disk0/00/01/61/00", "datestamp": "2023-06-12 18:06:11", "lastmod": "2023-11-08 00:43:31", "status_changed": "2023-06-12 18:06:11", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "schulte.sam.j@gmail.com", "id": "Schulte-Samuel-Jordan", "name": { "family": "Schulte", "given": "Samuel Jordan" }, "orcid": "0000-0001-9982-6504", "show_email": "YES" } ] }, "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", "ispublished": "unpub", "full_text_status": "public", "keywords": "hybridization chain reaction (HCR); dynamic nucleic acid nanotechnology; in situ hybridization (ISH); immunohistochemistry (IHC); immunofluorescence (IF); multiplexed imaging; spectral imaging with linear unmixing; 10-plex imaging; protein:protein complex imaging; quantitative HCR (qHCR); digital HCR (dHCR); lateral flow assay; rapid antigen test; SARS-CoV-2; COVID-19; nucleocapsid (N) protein", "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.
", "date": "2023", "date_type": "degree", "id_number": "CaltechTHESIS:06092023-193714022", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:06092023-193714022", "related_url": { "items": [ { "description": "Article adapted for Chapter 4", "type": "doi", "url": "https://doi.org/10.1021/acsinfecdis.2c00472" } ] }, "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "National Institutes of Health (NIBIB)", "grant_number": "R01EB006192" }, { "agency": "National Institutes of Health (NIGMS)", "grant_number": "GM008042" }, { "agency": "Richard N. Merkin Institute for Translational Research", "grant_number": "UNSPECIFIED" }, { "agency": "National Aeronautics and Space Administration (Translational Research Institute for Space Health)", "grant_number": "NNX16AO69A" }, { "agency": "Shurl and Kay Curci Foundation", "grant_number": "UNSPECIFIED" }, { "agency": "Beckman Institute (Programmable Molecular Technology Center)", "grant_number": "UNSPECIFIED" } ] }, "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Partial embargo requested. Full thesis and supplementary movie 2 withheld until 12 December 2023.", "deposited_by": "Samuel Schulte", "deposited_on": "2023-06-12 18:06:11", "doi": "10.7907/nzk8-2d38", "divisions": { "items": [ "div_bbe" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "woldb@caltech.edu", "id": "Wold-B-J", "name": { "family": "Wold", "given": "Barbara J." }, "orcid": "0000-0003-3235-8130", "role": "chair" }, { "email": "mbronner@caltech.edu", "id": "Bronner-M-E", "name": { "family": "Bronner", "given": "Marianne E." }, "orcid": "0000-0003-4274-1862", "role": "member" }, { "email": "luluqian@caltech.edu", "id": "Qian-Lulu", "name": { "family": "Qian", "given": "Lulu" }, "orcid": "0000-0003-4115-2409", "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2023-06-06", "gradofc_approval_date": "2023-06-12", "review_status": "approved", "option_major": { "items": [ "biology" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2023", "author_list": "Schulte, Samuel Jordan", "advisor_list": "Pierce, Niles A.", "comittee_list": "Wold, Barbara J.; Bronner, Marianne E.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/14456", "eprint_id": 14456, "rev_number": 60, "documents": [ { "id": "/id/document/135603", "doc_id": 135603, "rev_number": 5, "files": [ { "id": "/id/file/387645", "fileid": 387645, "datasetid": "document", "objectid": 135603, "filename": "thesis.pdf", "mime_type": "application/pdf", "hash": "3bd1b41deacc0b041315c7453ea36d02", "hash_type": "MD5", "filesize": 38787509, "mtime": "2021-12-22 13:24:18", "url": "/14456/3/thesis.pdf" } ], "eprint_id": 14456, "pos": 3, "placement": 3, "mime_type": "application/pdf", "format": "application/pdf", "format_desc": "Full thesis", "language": "en", "security": 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Computational approaches based on such models are central to current research directions across molecular programming, synthetic biology, and the life sciences more broadly. Our framework combines three ingredients. First, we develop new recursions to include contributions from coaxial and dangle stacking in an efficient and principled way. Second, we formulate the concept of an evaluation algebra, which defines the mathematical form of each subproblem in the dynamic program. Whereas previous modeling efforts have relied on case-by-case handling of different thermodynamic quantities, we use evaluation algebras to elegantly and efficiently compute a variety of physical quantities using the same recursions. Third, we develop efficient operation orders for a variety of physical quantities of experimental interest. Combining our advances, we are able to achieve speedups of 20-120x and scalable calculations of complexes of up to 30,000 nucleotides. Our achievements promise to dramatically expand the scope and utility of computational analysis and design of nucleic acid thermodynamics.
\r\n\r\nWhile current dynamic programming algorithms achieve efficient computation of thermodynamic quantities for a given nucleic acid sequence, they do not provide kinetic information. Therefore, investigations of secondary structure kinetics rely on stochastic simulations of trajectories in secondary structure space. We improve upon these simulation methodologies to achieve lower computational complexities and large empirical speedups. We extend our algorithms to an ensemble which fully includes coaxial and dangle stacking states, expanding the scope of the kinetic analysis that is currently possible.
\r\n\r\nCurrent secondary structure models are parametrized using thermodynamic information gleaned from decades of melt experiments of RNA and DNA in specific experimental conditions. Only rough kinetic information is currently available from past experiments, and information on solvent and material dependence is lacking. We develop a fully computational approach based on Gaussian processes and molecular dynamics in order to provide a generic method for estimating thermodynamic and kinetic parameters, applicable conceptually to any nucleic acid material and experimental setting of interest. Our methodology offers an atomistic view of nucleic acid base pairing and faithfully reproduces most experimental data. It thus provides a powerful black-box approach for extensibly calculating the kinetic and thermodynamic parameters that secondary structure models require.
", "date": "2022", "date_type": "degree", "id_number": "CaltechTHESIS:12212021-202554260", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:12212021-202554260", "related_url": { "items": [ { "description": "Article adapted for Chapter II and Appendix A", "type": "doi", "url": "https://doi.org/10.1021/acssynbio.9b00523" } ] }, "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Melissa Ray", "suggestions": "2022-05-18 - Full thesis fully embargoed until 1 December 2022. Appeal approved after committee review. // Appeal denied 2-022-0504. Author and advisor notified 2022-05-16 by KJ //\r\n//Appeal of denial received 11Feb2022. Continue embargo until committee has responded. //\r\n\r\n// First embargo request denied 2021-01-15. Kathy is notifying student and and pointing out appeal procedure. SKJ 2021-28-01 // Withheld until 2022-06-22 // From author: This thesis contains sensitive material that is currently being finalized to submit to a peer-reviewed journal. Chapters III and IV are planned to be prepared and edited as a single paper submitted to ACS Synthetic Biology,considered a high-profile journal in synthetic biology. (The ACS submission journal policy for submission embargoes are \"Authors may reuse all or part of the Submitted, Accepted or Published Work in a thesis or dissertation that the Author writes and is required to submit to satisfy the criteria of degree-granting institutions\".) Final simulations for DNA must be performed, supplementary information prepared, and the text must be revised and reorganized. We estimate these tasks to take 4-6 months. Final simulations should be finished within 2 months, an initial draft within 3 months, and a revised draft within 4 months before final submission and review.", "deposited_by": "Mark Fornace", "deposited_on": "2022-01-28 17:03:53", "doi": "10.7907/ayeg-at42", "divisions": { "items": [ "div_chem" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "tfm@caltech.edu", "id": "Miller-T-F", "name": { "family": "Miller", "given": "Thomas F." }, "orcid": "0000-0002-1882-5380", "role": "chair" }, { "email": "garnetc@caltech.edu", "id": "Chan-G-K", "name": { "family": "Chan", "given": "Garnet K." }, "orcid": "0000-0001-8009-6038", "role": "member" }, { "email": "zgw@caltech.edu", "id": "Wang-Zhen-Gang", "name": { "family": "Wang", "given": "Zhen-Gang" }, "orcid": "0000-0002-3361-6114", "role": "member" }, { "email": "winfree@caltech.edu", "id": "Winfree-E", "name": { "family": "Winfree", "given": "Erik" }, "orcid": "0000-0002-5899-7523", "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2021-12-07", "gradofc_approval_date": "2022-01-27", "review_status": "approved", "option_major": { "items": [ "chemistry" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. 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"Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function via Dynamic RNA Nanotechnology", "ispublished": "unpub", "full_text_status": "public", "keywords": "Small conditional RNA (scRNA); programmable conditional regulators; allosteric regulators; CRISPR/Cas; dynamic RNA nanotechnology; molecular programming; rational design; gene regulation; Cas9; conditional guide RNA; synthetic biology", "abstract": "A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. There are two possible logical directions for single-input cgRNAs: constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON\u2192OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF\u2192ON logic). Using an in vitro assay for cgRNA activity with synthetic trigger, in vitro transcribed cgRNA, and recombinant dCas9, we observe a conditional (ON\u2192OFF logic) response for a set of four allosteric constitutively active cgRNAs with a median \u22486% crosstalk between noncognate cgRNA/trigger pairs. Motivated by the observed lack of conditional response of this mechanism when ported to E. coli, we describe a systematic study of unstructured sequence inserts into the standard gRNA structure and report the conditional response of a set of 34 candidate cgRNAs in living cells. Molecular mechanisms for both ON\u2192OFF and OFF\u2192ON cgRNAs are demonstrated in E. coli. For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to produce an orthogonal library of cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of \u224815-fold for a library of three orthogonal ON\u2192OFF \"splinted switch\" cgRNA/trigger pairs, and \u22483-fold for a library of three orthogonal OFF\u2192ON \"toehold switch\" cgRNA/trigger pairs; the median crosstalk within each library is <2% and \u224820% for the two mechanisms, respectively. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for conditional gene regulation and synthetic biology.
", "date": "2020", "date_type": "degree", "id_number": "CaltechTHESIS:11042019-135312842", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:11042019-135312842", "related_url": { "items": [ { "description": "Article content incorporated into Chapters 4 and 5, supporting information content incorporated into Chapters 3-5 and Appendices", "type": "doi", "url": "https://doi.org/10.1021/acscentsci.9b00340" } ] }, "referencetext": { "items": [ "1. Kallenbach, N. R., Ma, R.-I. & Seeman, N. C. An immobile nucleic acid junction constructed from oligonucleotides. Nature 305, 829\u2013831 (1983).\r\n2. Chen, J. & Seeman, N. C. Synthesis from DNA of a molecule with the connectivity of a cube. Nature 350, 631\u2013633 (1991).\r\n3. Winfree, E., Liu, F., Wenzler, L. A. & Seeman, N. C. Design and self-assembly of two-dimensional DNA crystals. Nature 394, 539\u2013544 (1998).\r\n4. Seeman, N. C. & Sleiman, H. F. DNA nanotechnology. Nat. Rev. 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Nature 403, 335\u2013338 (2000)." ] }, "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Melissa Ray", "deposited_by": "Mikhail Hanewich Hollatz", "deposited_on": "2019-11-05 19:20:23", "doi": "10.7907/NS2B-DJ96", "divisions": { "items": [ "div_bbe" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "winfree@caltech.edu", "id": "Winfree-E", "name": { "family": "Winfree", "given": "Erik" }, "orcid": "0000-0002-5899-7523", "role": "chair" }, { "email": "elowitz@caltech.edu", "id": "Elowitz-M-B", "name": { "family": "Elowitz", "given": "Michael B." }, "orcid": "0000-0002-1221-0967", "role": "member" }, { "email": "murray@cds.caltech.edu", "id": "Murray-R-M", "name": { "family": "Murray", "given": "Richard M." }, "orcid": "0000-0002-5785-7481", "role": "member" }, { "email": "mikhail@caltech.edu", "id": "Shapiro-M-G", "name": { "family": "Shapiro", "given": "Mikhail G." }, "orcid": "0000-0002-0291-4215", "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2019-06-19", "gradofc_approval_date": "2019-11-05", "review_status": "approved", "option_major": { "items": [ "bioeng" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. 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"disk0/00/01/18/08", "datestamp": "2019-10-25 00:26:08", "lastmod": "2023-11-08 00:43:31", "status_changed": "2019-10-25 00:26:08", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "njpcaltech@gmail.com", "id": "Porubsky-Nicholas-James", "name": { "family": "Porubsky", "given": "Nicholas James" }, "orcid": "0000-0001-6330-2645", "show_email": "YES" } ] }, "title": "Enhanced Algorithms for Analysis and Design of Nucleic Acid Reaction Pathways", "ispublished": "unpub", "full_text_status": "public", "keywords": "nucleic acids; DNA; RNA; test tube; reaction pathway; secondary structure; multistate test tube design; coaxial and dangle stacking;", "abstract": "Nucleic acids provide a powerful platform for programming at the molecular level. This is possible because the free energy of nucleic acid structures is dominated by the local interactions of base pairing and base pair stacking. The nearest neighbor secondary structure model implied by these energetics has enabled development of a set of algorithms for calculating thermodynamic quantities of nucleic acid sequences. Molecular programmers and synthetic biologists continue to extend their reach to larger, more complicated nucleic acid complexes, reaction pathways, and systems. This necessitates a focus on new algorithm development and efficient implementations to enable analysis and design of such systems.
\r\n\r\nConcerning analysis of nucleic acids, we collect seemingly diverse algorithms under a unified three-component dynamic programming framework consisting of: 1) recursions that specify the dependencies between subproblems and incorporate the details of the structural ensemble and the free energy model, 2) evaluation algebras that define the mathematical form of each subproblem, 3) operation orders that specify the computational trajectory through the dependency graph of subproblems. Changes to the set of recursions allows operation over the complex ensemble including coaxial and dangle stacking states, affecting all thermodynamic quantities. An updated operation order for structure sampling allows simultaneous generation of a set of structures sampled from the Boltzmann distribution in time that scales empirically sublinearly in the number of samples and leads to an order of magnitude or more speedup over repeated single-structure sampling.
\r\n\r\nFor the problem of sequence design for reaction pathway engineering, we introduce an optimization algorithm to minimize the multitstate test tube ensemble defect, which simultaneously designs for reactant, intermediate, and product states along the reaction pathway (positive design) and against crosstalk interactions (negative design). Each of these on-pathway or crosstalk states is represented as a target test tube ensemble containing arbitrary numbers of on-target complexes, each with a target secondary structure and target concentration, and arbitrary numbers of off-target complexes, each with vanishing target concentration. Our test tube specification formalism enables conversion of a reaction pathway specification into a set of target test tubes. Sequences are designed subject to a set of hard constraints allowing specification of properties such as sequence composition, sequence complementarity, prevention of unwanted sequence patterns, and inclusion of biological sequences. We then extend this algorithm with soft constraints, enhancing flexibility through new constraint types and reducing design cost by up to two orders of magnitude in the most highly constrained cases. These soft constraints enable multiobjective design of the multitstate test tube ensemble defect simultaneously with heuristics for avoiding kinetic traps and equalizing reaction rates to further aid reaction pathway engineering.
\r\n", "date": "2020", "date_type": "degree", "id_number": "CaltechTHESIS:10062019-213347699", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:10062019-213347699", "related_url": { "items": [ { "description": "article adapted for ch. 3", "type": "doi", "url": "https://doi.org/10.1021/jacs.6b12693" } ] }, "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "National Science Foundation", "grant_number": "Software Elements NSF-OAC-1835414" }, { "agency": "National Science Foundation", "grant_number": "INSPIRE NSF-CHE-1643606" }, { "agency": "National Science Foundation", "grant_number": "Molecular Programming Project NSF-CCF-1317694" }, { "agency": "National Institutes of Health", "grant_number": "National Research Service Award T32 GM007616" }, { "agency": "Programmable Molecular Technology Center (PMTC) within the Beckman Institute at Caltech" }, { "agency": "AWS/IST Cloud Credit Program at Caltech" }, { "agency": "Microsoft Azure sponsorship" } ] }, "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Released to public view 16 October 2020. //\r\n// Withheld until 16 October 2020. 1st Embargo extension request approved 4/16/2020. From author: We are working on resubmitting the following paper, and that will not be finished before the embargo expiration on April 24, 2019: Journal: ACS Synthetic Biology; Manuscript ID: sb-2019-00523y; Title: \"A Unified Dynamic Programming Framework for the Analysis of Interacting Nucleic Acid Strands: Enhanced Models, Scalability, and Speed;\" Author(s): Fornace, Mark; Porubsky, Nicholas; Pierce, Niles.\r\n\r\n// Original embargo request // Withheld until 24 April 2020. Student submitting the paper to an ACS journal (ACS Synthetic Biology) - 10/08/2019 //", "deposited_by": "Nicholas Porubsky", "deposited_on": "2019-10-25 00:26:08", "doi": "10.7907/M8CZ-MW98", "divisions": { "items": [ "div_chem" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "winfree@caltech.edu", "id": "Winfree-E", "name": { "family": "Winfree", "given": "Erik" }, "role": "chair" }, { "email": "zgw@cheme.caltech.edu", "id": "Wang-Zhen-Gang", "name": { "family": "Wang", "given": "Zhen-Gang" }, "role": "member" }, { "email": "rustem@caltech.edu", "id": "Ismagilov-R-F", "name": { "family": "Ismagilov", "given": "Rustem F." }, "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2019-09-16", "gradofc_approval_date": "2019-10-24", "review_status": "approved", "option_major": { "items": [ "chemeng" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2020", "author_list": "Porubsky, Nicholas James", "advisor_list": "Pierce, Niles A.", "comittee_list": "Winfree, Erik; Wang, Zhen-Gang; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/9835", "eprint_id": 9835, "rev_number": 24, "documents": [ { "id": "/id/document/70062", "doc_id": 70062, "rev_number": 4, "files": [ { "id": "/id/file/191726", "fileid": 191726, "datasetid": "document", "objectid": 70062, "filename": "Thesis Final v2.pdf", "mime_type": "application/pdf", "hash": "e9fdd947479da8e5226b418093ff833e", "hash_type": "MD5", "filesize": 75628401, "mtime": "2016-06-03 21:40:24", "url": "/9835/1/Thesis Final v2.pdf" } ], "eprint_id": 9835, "pos": 1, "placement": 1, "mime_type": "application/pdf", "format": "application/pdf", "language": "en", "security": "public", "license": "other", 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"http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/70062" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/70062" } ] } } ], "eprint_status": "archive", "userid": 10803, "dir": "disk0/00/00/98/35", "datestamp": "2016-06-06 20:21:50", "lastmod": "2023-11-08 00:43:32", "status_changed": "2016-06-06 20:21:50", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "aneesh@outlook.com", "id": "Acharya-Aneesh", "name": { "family": "Acharya", "given": "Aneesh" }, "orcid": "0000-0002-4402-7147", "show_email": "YES" } ] }, "title": "Multiplexed Analysis of Diverse RNA Classes via Hybridization Chain Reaction", "ispublished": "unpub", "full_text_status": "public", "keywords": "hybridization chain reaction; molecular programming; in situ hybridization; microRNA; bacteria mouse colon; flow cytometry; spectral imaging", "abstract": "Gene circuits are complex biological networks composed of numerous regulatory elements, including transcription factors, mRNAs, and microRNAs. Fluorescent in situ hybridization (FISH) is a powerful method for spatially mapping expression levels of RNA elements within an intact organism, but traditional methods exhibit at least one of the following drawbacks: low signal-to-background, arduous and/or destructive multiplexing, and non-quantitative signal. These issues are all overcome using in situ amplification based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger the self-assembly of fluorophore-labeled nucleic acid hairpins into tethered fluorescent amplification polymers. In situ HCR enables straightforward multiplexing, high signal-to-background, and quantitative signal. Here, we address three key scenarios in which HCR enables novel applications for in situ hybridization. First, we address the challenge of sorting cell subpopulations based on mRNA abundance using flow cytometry to enable high-throughput measurement of the signal intensity from individual cells. High signal is required to overcome the background autofluorescence integrated over the volume of each cell. Quantitative HCR signal amplification enables multi-dimensional sorting of mammalian cell lines based on expression levels of multiple target mRNAs. Second, we address the challenge of mapping multiple microRNA and mRNA targets simultaneously. Traditional methods enable mapping of single microRNA targets in isolation and use costly LNA probes with proprietary compositions that differ for each target. Here we develop in situ HCR for multiplexed mapping not only of microRNAs, but of microRNAs and mRNAs together, using non-proprietary 2'OMe-RNA probes for miRNA targets and DNA probes for mRNA targets. Third, to enable studies of gut flora, we address the challenge of mapping spatial relationships between different bacterial species within the intact mouse colon. In situ HCR enables multiplexed discrimination of multiple closely-related Bacteroides species with rRNAs that differ by only a few nucleotides. In summary, this thesis presents in situ HCR as a tool for multiplexed analysis of diverse RNA classes and expands the range of gene circuit regulatory elements that can be spatially and quantitatively mapped.", "date": "2016", "date_type": "degree", "id_number": "CaltechTHESIS:06032016-143422651", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:06032016-143422651", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Restricted to campus until 6 June 2017. // Released to public view 21 December 2017.", "deposited_by": "Aneesh Acharya", "deposited_on": "2016-06-06 20:21:50", "doi": "10.7907/Z95M63N0", "divisions": { "items": [ "div_bbe" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "melowitz@caltech.edu", "id": "Elowitz-M-B", "name": { "family": "Elowitz", "given": "Michael B." }, "role": "chair" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" }, { "email": "sfraser@provost.USC.edu", "id": "Fraser-S-E", "name": { "family": "Fraser", "given": "Scott E." }, "role": "member" }, { "email": "sarkis@caltech.edu", "id": "Mazmanian-S-K", "name": { "family": "Mazmanian", "given": "Sarkis K." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2016-05-19", "gradofc_approval_date": "2016-06-06", "review_status": "approved", "option_major": { "items": [ "bioeng" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. 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"items": [ { "email": "nhusain@gmail.com", "id": "Husain-Naeem-Shahab", "name": { "family": "Husain", "given": "Naeem Shahab" }, "show_email": "NO" } ] }, "title": "Mapping mRNA and Protein Expression with High Signal-to-Background in Diverse Organisms", "ispublished": "unpub", "full_text_status": "public", "keywords": "hybridization chain reaction, in situ hybridization, immunohistochemistry", "note": "The Caltech Option \"Biochemistry and Molecular Biophysics\" is considered an Interdisciplinary Program. The author studied in both the Biology and Biological Engineering (BBE) and the Chemistry and Chemical Engineering (CCE) divisions to earn her degree. He is listed under CCE in the 2016 Commencement program.", "abstract": "In situ hybridization (ISH) techniques allow for the study of the nucleic acid expression within whole biological samples. The quality of probes for ISH dictates how accurate and bright the signal is for the experiment; however, there is currently not a systematic way to determine what the best probe set would be. In response to this, we have developed a framework to optimize an ISH probe set to achieve the greatest signal-to-background ratio. As methods like ISH help obtain more information about biological processes, there is a growing desire to simultaneously analyze various targets within the same sample to examine these complex genetic interactions. To facilitate this, a novel amplification technique called hybridization chain reaction (HCR) has allowed for the in situ detection of multiple target mRNAs concurrently in zebrafish embryos. We have now expanded this technology further by adapting HCR amplification for ISH to other model organisms, particularly, whole mount Drosophila melanogaster embryos and formalin-fixed parafin-embedded human tissue sections. Beyond looking at mRNA, immunohistochemistry (IHC) provides another tool to understand biological systems by analyzing protein expression patterns. The ability to easily look at both mRNAs and proteins in the same sample offers significant advantages as each provides unique information, but current methods are technically difficult and labor intensive. In response, we have engineered a scheme to use HCR to amplify signal for IHC. We then used this advancement to develop a straightforward protocol using HCR amplification for simultaneous detection of multiple proteins and mRNAs with a high signal-to-background ratio.", "date": "2016", "date_type": "degree", "id_number": "CaltechTHESIS:02252016-123239637", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:02252016-123239637", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "NIH", "grant_number": "NAP-HCRENEWSCAPII-1-NIH.HCRENEW" }, { "agency": "NSF Graduate Fellowship" } ] }, "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Restricted to Caltech community only until 1 March 2019. \r\n // Under secondary review, do not release. 03232018 // Originally Withheld until 31 January 2017. // 03082017: Released and Restricted to Caltech community only until 1 September 2017. // Extension requested 12/8/2017 ; sent to review committee.", "deposited_by": "Naeem Husain", "deposited_on": "2017-03-08 21:17:50", "doi": "10.7907/Z9DF6P73", "divisions": { "items": [ "div_chem" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "lcai@caltech.edu", "id": "Cai-Long", "name": { "family": "Cai", "given": "Long" }, "role": "chair" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" }, { "email": "dprober@caltech.edu", "id": "Prober-D-A", "name": { "family": "Prober", "given": "David A." }, "role": "member" }, { "email": "pws@caltech.edu", "id": "Sternberg-P-W", "name": { "family": "Sternberg", "given": "Paul W." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2015-12-16", "gradofc_approval_date": "2016-02-29", "review_status": "approved", "option_major": { "items": [ "biochem" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2016", "author_list": "Husain, Naeem Shahab", "advisor_list": "Pierce, Niles A.", "comittee_list": "Cai, Long; Pierce, Niles A.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/8917", "eprint_id": 8917, "rev_number": 35, "documents": [ { "id": "/id/document/57518", "doc_id": 57518, "rev_number": 5, "files": [ { "id": "/id/file/163125", "fileid": 163125, "datasetid": "document", "objectid": 57518, "filename": "VikasTrivedi_2015_Thesis.pdf", "mime_type": "application/pdf", "hash": "4062cfa57db070b943ab28681b44ba63", "hash_type": "MD5", "filesize": 112953202, "mtime": "2015-06-01 18:50:39", "url": "/8917/1/VikasTrivedi_2015_Thesis.pdf" } ], "eprint_id": 8917, "pos": 1, "placement": 1, "mime_type": "application/pdf", "format": "application/pdf", "language": "en", "security": "public", "license": 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"media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/57518" }, { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/57518" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/57518" } ] } } ], "eprint_status": "archive", "userid": 5291, "dir": "disk0/00/00/89/17", "datestamp": "2016-01-19 22:00:18", "lastmod": "2023-11-08 18:28:33", "status_changed": "2016-01-19 22:00:18", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "viktri.iitk@gmail.com", "id": "Trivedi-Vikas", "name": { "family": "Trivedi", "given": "Vikas" }, "orcid": "0000-0003-0953-0553", "show_email": "YES" } ] }, "title": "From Molecules to Organs : Microscopy and Multi-Scale Nature of Development", "ispublished": "unpub", "full_text_status": "public", "keywords": "Developmental biology, 2-photon light sheet, Hybridization Chain Reaction, Cardiac development, Cartilage development", "abstract": "Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development. ", "date": "2015", "date_type": "degree", "id_number": "CaltechTHESIS:05292015-080950114", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:05292015-080950114", "related_url": { "items": [ { "description": "Article adapted for ch. 4", "type": "doi", "url": "http://dx.doi.org/ncomms7798" }, { "description": "Article adapted for ch. 7", "type": "doi", "url": "http://dx.doi.org/10.1364/BOE.6.002056" }, { "description": "Article adapted for ch. 8", "type": "doi", "url": "http://dx.doi.org/10.1016/j.bpj.2012.11.1140" } ] }, "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Restricted to Caltech community only until 6 June 2020. Advisor ok to release 12/8/2017.\r\n", "deposited_by": "Vikas Trivedi", "deposited_on": "2016-01-19 22:00:18", "doi": "10.7907/Z9SX6B59", "divisions": { "items": [ "div_bbe" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "sfraser@provost.usc.edu", "id": "Fraser-S-E", "name": { "family": "Fraser", "given": "Scott E." }, "orcid": "0000-0002-5377-0223", "role": "co-advisor" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "co-advisor" } ] }, "thesis_committee": { "items": [ { "email": "ravi@caltech.edu", "id": "Ravichandran-G", "name": { "family": "Ravichandran", "given": "Guruswami" }, "orcid": "0000-0002-2912-0001", "role": "chair" }, { "email": "mgharib@caltech.edu", "id": "Gharib-M", "name": { "family": "Gharib", "given": "Morteza" }, "orcid": "0000-0003-0754-4193", "role": "member" }, { "email": "phillips@pboc.caltech.edu", "id": "Phillips-R-B", "name": { "family": "Phillips", "given": "Robert B." }, "orcid": "0000-0003-3082-2809", "role": "member" }, { "email": "sfraser@provost.usc.edu", "id": "Fraser-S-E", "name": { "family": "Fraser", "given": "Scott E." }, "orcid": "0000-0002-5377-0223", "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "orcid": "0000-0003-2367-4406", "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2015-05-19", "gradofc_approval_date": "2015-06-04", "review_status": "approved", "option_major": { "items": [ "bioeng" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. 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"public", "keywords": "DNA;RNA;nucleic acid design;RNA folding;DNA folding;DNA design;inverse folding;molecular programming", "abstract": "Nucleic acids are a useful substrate for engineering at the molecular level. Designing the detailed energetics and kinetics of interactions between nucleic acid strands remains a challenge. Building on previous algorithms to characterize the ensemble of dilute solutions of nucleic acids, we present a design algorithm that allows optimization of structural features and binding energetics of a test tube of interacting nucleic acid strands. We extend this formulation to handle multiple thermodynamic states and combinatorial constraints to allow optimization of pathways of interacting nucleic acids. In both design strategies, low-cost estimates to thermodynamic properties are calculated using hierarchical ensemble decomposition and test tube ensemble focusing. These algorithms are tested on randomized test sets and on example pathways drawn from the molecular programming literature. To analyze the kinetic properties of designed sequences, we describe algorithms to identify dominant species and kinetic rates using coarse-graining at the scale of a small box containing several strands or a large box containing a dilute solution of strands.", "date": "2014", "date_type": "degree", "id_number": "CaltechTHESIS:03272014-193400848", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:03272014-193400848", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Restricted to Caltech community only until 6 May 2020.", "deposited_by": "Brian Wolfe", "deposited_on": "2014-05-29 21:39:18", "doi": "10.7907/G4XZ-ZF47", "divisions": { "items": [ "div_bbe" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "winfree@caltech.edu", "id": "Winfree-E", "name": { "family": "Winfree", "given": "Erik" }, "role": "chair" }, { "email": "murray@cds.caltech.edu", "id": "Murray-R-M", "name": { "family": "Murray", "given": "Richard M." }, "role": "member" }, { "email": "tfm@caltech.edu", "id": "Miller-T-F", "name": { "family": "Miller", "given": "Thomas F." }, "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2014-05-06", "gradofc_approval_date": "2014-05-28", "review_status": "approved", "option_major": { "items": [ "bioeng" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. 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"unpub", "full_text_status": "public", "keywords": "conditional RNAi, small conditional RNA, nucleic acid engineering, hybridization cascades, nucleic acid logic gates", "abstract": "RNA interference (RNAi) is a powerful biological pathway allowing for sequence-specific knockdown of any gene of interest. While RNAi is a proven tool for probing gene function in biological circuits, it is limited by being constitutively ON and executes the logical operation: silence gene Y. To provide greater control over post-transcriptional gene silencing, we propose engineering a biological logic gate to implement \u201cconditional RNAi.\u201d Such a logic gate would silence gene Y only upon the expression of gene X, a completely unrelated gene, executing the logic: if gene X is transcribed, silence independent gene Y. Silencing of gene Y could be confined to a specific time and/or tissue by appropriately selecting gene X.
\r\n\r\nTo implement the logic of conditional RNAi, we present the design and experimental validation of three nucleic acid self-assembly mechanisms which detect a sub-sequence of mRNA X and produce a Dicer substrate specific to gene Y. We introduce small conditional RNAs (scRNAs) to execute the signal transduction under isothermal conditions. scRNAs are small RNAs which change conformation, leading to both shape and sequence signal transduction, in response to hybridization to an input nucleic acid target. While all three conditional RNAi mechanisms execute the same logical operation, they explore various design alternatives for nucleic acid self-assembly pathways, including the use of duplex and monomer scRNAs, stable versus metastable reactants, multiple methods of nucleation, and 3-way and 4-way branch migration.
\r\n\r\nWe demonstrate the isothermal execution of the conditional RNAi mechanisms in a test tube with recombinant Dicer. These mechanisms execute the logic: if mRNA X is detected, produce a Dicer substrate targeting independent mRNA Y. Only the final Dicer substrate, not the scRNA reactants or intermediates, is efficiently processed by Dicer. Additional work in human whole-cell extracts and a model tissue-culture system delves into both the promise and challenge of implementing conditional RNAi in vivo.
", "date": "2013", "date_type": "degree", "id_number": "CaltechTHESIS:06072013-160243112", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:06072013-160243112", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Released to public view 23 October 2020. //\r\n// Restricted to Caltech community only until 6 June 2020.", "deposited_by": "Lisa Hochrein", "deposited_on": "2016-01-19 21:57:50", "doi": "10.7907/NTZT-8Q67", "divisions": { "items": [ "div_chem" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "datirrell@caltech.edu", "id": "Tirrell-D-A", "name": { "family": "Tirrell", "given": "David A." }, "role": "chair" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" }, { "email": "mdavis@cheme.caltech.edu", "id": "Davis-M-E", "name": { "family": "Davis", "given": "Mark E." }, "role": "member" }, { "email": "JRossi@coh.org", "id": "Rossi-J-J", "name": { "family": "Rossi", "given": "John J." }, "role": "member" }, { "email": "jburnett@coh.org", "id": "Burnett-J", "name": { "family": "Burnett", "given": "John" }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2013-05-31", "gradofc_approval_date": "2013-06-11", "review_status": "approved", "option_major": { "items": [ "chemeng" ] }, "option_minor": { "items": [ "biology" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2013", "author_list": "Hochrein, Lisa Marie", "advisor_list": "Pierce, Niles A.", "comittee_list": "Tirrell, David A.; Pierce, Niles A.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/7874", "eprint_id": 7874, "rev_number": 28, "documents": [ { "id": "/id/document/41469", "doc_id": 41469, "rev_number": 7, "files": [ { "id": "/id/file/125366", "fileid": 125366, "datasetid": "document", "objectid": 41469, "filename": "Sternberg_Jonathan_2013_thesis.pdf", "mime_type": "application/pdf", "hash": "be6e74e2cbe2bb4e3c8dd42518c8e42a", "hash_type": "MD5", "filesize": 63369791, "mtime": "2013-06-08 00:16:19", "url": "/7874/1/Sternberg_Jonathan_2013_thesis.pdf" } ], "eprint_id": 7874, "pos": 1, "placement": 1, "mime_type": "application/pdf", "format": "application/pdf", "language": "en", "security": 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"main": "indexcodes.txt", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/41469" }, { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/41469" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/41469" } ] } } ], "eprint_status": "archive", "userid": 9771, "dir": "disk0/00/00/78/74", "datestamp": "2016-01-19 21:56:47", "lastmod": "2023-11-08 00:43:32", "status_changed": "2016-01-19 21:56:47", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "sternbergj@gmail.com", "id": "Sternberg-Jonathan-Ben-Zion", "name": { "family": "Sternberg", "given": "Jonathan Ben-Zion" }, "show_email": "NO" } ] }, "title": "Signal Transduction with Hybridization Chain Reactions", "ispublished": "unpub", "full_text_status": "public", "keywords": "diagnostic; diagnostics; triggered; amplification; isothermal; enzyme-free", "abstract": "Some of the most exciting developments in the field of nucleic acid engineering include the utilization of synthetic nucleic acid molecular devices as gene regulators, as disease marker detectors, and most recently, as therapeutic agents. The common thread between these technologies is their reliance on the detection of specific nucleic acid input markers to generate some desirable output, such as a change in the copy number of an mRNA (for gene regulation), a change in the emitted light intensity (for some diagnostics), and a change in cell state within an organism (for therapeutics). The research presented in this thesis likewise focuses on engineering molecular tools that detect specific nucleic acid inputs, and respond with useful outputs.
\r\n\r\nFour contributions to the field of nucleic acid engineering are presented: (1) the construction of a single nucleotide polymorphism (SNP) detector based on the mechanism of hybridization chain reaction (HCR); (2) the utilization of a single-stranded oligonucleotide molecular Scavenger as a means of enhancing HCR selectivity; (3) the implementation of Quenched HCR, a technique that facilitates transduction of a nucleic acid chemical input into an optical (light) output, and (4) the engineering of conditional probes that function as sequence transducers, receiving target signal as input and providing a sequence of choice as output. These programmable molecular systems are conceptually well-suited for performing wash-free, highly selective rapid genotyping and expression profiling in vitro, in situ, and potentially in living cells.
", "date": "2013", "date_type": "degree", "id_number": "CaltechTHESIS:06072013-170244348", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:06072013-170244348", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Restricted to Caltech community only until 6 June 2020. // Released 03092017 with advisor approval.", "deposited_by": "Jonathan Sternberg", "deposited_on": "2016-01-19 21:56:47", "doi": "10.7907/Z90Z719Z", "divisions": { "items": [ "div_eng" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles/ A" }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "tirrell@caltech.edu", "id": "Tirrell-D-A", "name": { "family": "Tirrell", "given": "David A." }, "role": "chair" }, { "email": "scott.fraser@usc.edu", "id": "Fraser-S-E", "name": { "family": "Fraser", "given": "Scott E." }, "role": "member" }, { "email": "melowitz@caltech.edu", "id": "Elowitz-M-B", "name": { "family": "Elowitz", "given": "Michael B." }, "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2013-05-22", "gradofc_approval_date": "2013-06-11", "review_status": "approved", "option_major": { "items": [ "bioeng" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2013", "author_list": "Sternberg, Jonathan Ben-Zion", "advisor_list": "Pierce, Niles/ A", "comittee_list": "Tirrell, David A.; Fraser, Scott E.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/7733", "eprint_id": 7733, "rev_number": 61, "documents": [ { "id": "/id/document/38658", "doc_id": 38658, "rev_number": 8, "files": [ { "id": "/id/file/119657", "fileid": 119657, "datasetid": "document", "objectid": 38658, "filename": "MS_thesis_chapter1.pdf", "mime_type": "application/pdf", "hash": "444aa688540f7412553ed278d55fc98e", "hash_type": "MD5", "filesize": 337320, "mtime": "2013-05-23 16:21:02", "url": "/7733/7/MS_thesis_chapter1.pdf" } ], "eprint_id": 7733, "pos": 7, "placement": 2, "mime_type": "application/pdf", "format": "application/pdf", "format_desc": "Chapter 1", "language": "en", 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{ "family": "Schwarzkopf", "given": "Ma'ayan" }, "show_email": "NO" } ] }, "title": "Engineering Nucleic Acid Mechanisms for Regulation and Readout of Gene Expression: Conditional Dicer Substrate Formation and Sensitive Multiplexed Northern Blots", "ispublished": "unpub", "full_text_status": "public", "keywords": "nucleic acid engineering; RNAi; RNA interference; northern blot; hybridization chain reaction; HCR; nanotechnology; conditional RNAi; scRNA; scDNA", "abstract": "Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.
\r\n\r\nRNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.
\r\n \r\nSignal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.
\r\n", "date": "2013", "date_type": "degree", "id_number": "CaltechTHESIS:05212013-161536447", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:05212013-161536447", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Restricted to Caltech community only until 6 June 2020. Released 03092017 with advisor approval.", "deposited_by": "Maayan Schwarzkopf", "deposited_on": "2016-01-19 22:08:20", "doi": "10.7907/Z94Q7S0V", "divisions": { "items": [ "div_biol" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "deshaies@caltech.edu", "id": "Deshaies-R-J", "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "role": "chair" }, { "email": "baltimo@caltech.edu", "id": "Baltimore-D-L", "name": { "family": "Baltimore", "given": "David L." }, "role": "member" }, { "email": "haybruce@caltech.edu", "id": "Hay-B-A", "name": { "family": "Hay", "given": "Bruce A." }, "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2013-05-09", "gradofc_approval_date": "2013-05-29", "review_status": "approved", "option_major": { "items": [ "biology" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2013", "author_list": "Schwarzkopf, Ma'ayan", "advisor_list": "Pierce, Niles A.", "comittee_list": "Deshaies, Raymond Joseph; Baltimore, David L.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/5488", "eprint_id": 5488, "rev_number": 29, "documents": [ { "id": "/id/document/9165", "doc_id": 9165, "rev_number": 6, "files": [ { "id": "/id/file/57559", "fileid": 57559, "datasetid": "document", "objectid": 9165, "filename": "HarryChoi_Thesis_submit.pdf", "mime_type": "application/pdf", "filesize": 70867126, "mtime": "2012-12-26 03:20:40", "url": "/5488/1/HarryChoi_Thesis_submit.pdf" } ], "eprint_id": 5488, "pos": 1, "mime_type": "application/pdf", "format": "application/pdf", "format_desc": "Complete Thesis", "language": "en", "security": "public", "license": "other", "main": 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"language": "en", "security": "public", "license": "other", "main": "indexcodes.txt", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/9165" }, { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/9165" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/9165" } ] } }, { "id": "/id/document/125510", "doc_id": 125510, "rev_number": 1, "files": [ { "id": "/id/file/370944", "fileid": 370944, "datasetid": "document", "objectid": 125510, "filename": "indexcodes.txt", "mime_type": "text/plain", "hash": "140ec93c784ced3be0045909b9ec2684", "hash_type": "MD5", "filesize": 787, "mtime": "2021-06-24 00:13:13", "url": "/5488/7/indexcodes.txt" } ], "eprint_id": 5488, "pos": 7, "placement": 7, "mime_type": "text/plain", "format": "other", "format_desc": "Generate index codes conversion from text/plain to indexcodes", "language": "en", "security": "internal", "license": "other", "main": "indexcodes.txt", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/11948" }, { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/11948" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/11948" } ] } } ], "eprint_status": "archive", "userid": 678, "dir": "disk0/00/00/54/88", "datestamp": "2011-01-25 00:26:57", "lastmod": "2023-11-08 00:43:32", "status_changed": "2011-01-25 00:26:57", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "hmtchoi@gmail.com", "id": "Choi-Harry-Ming-Tak", "name": { "family": "Choi", "given": "Harry Ming Tak" }, "orcid": "0000-0002-1530-0773", "show_email": "YES" } ] }, "title": "Programmable In Situ Amplification for Multiplexed Bioimaging\r ", "ispublished": "unpub", "full_text_status": "public", "keywords": "Nucleic Acids; DNA; RNA; In Situ Hybridization; Amplification; Hybridization Chain Reaction; HCR; Multiplexing; Zebrafish; Motor; Autonomous Walker", "abstract": "In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously detect multiple target mRNAs in vertebrate embryos and tissue sections \u2013 a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. This thesis presents a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reaction (HCR). Using this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. Robust performance and high signal-to-background are achieved when imaging five target mRNAs at the same time in fixed whole-mount zebrafish embryos. The programmability and sequence specificity of these amplification cascades enable all five amplifiers to operate orthogonally at the same time in the same sample. The fact that amplification polymers are triggered to self-assemble in situ results in excellent sample penetration and high signal-to-background. These properties suggest the broad applicability of fluorescent in situ HCR amplification to multiplexed imaging of mRNA expression in normal and pathological cells, embryos, and tissue sections.\r\n", "date": "2010", "date_type": "degree", "id_number": "CaltechTHESIS:12292009-170615072", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:12292009-170615072", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Gayle Hammer", "deposited_by": "Harry Choi", "deposited_on": "2011-01-25 00:26:57", "doi": "10.7907/K4A3-1K41", "divisions": { "items": [ "div_eng" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "sefraser@caltech.edu", "id": "Fraser-S-E", "name": { "family": "Fraser", "given": "Scott E." }, "role": "chair" }, { "email": "phillips@pboc.caltech.edu", "id": "Phillips-R-B", "name": { "family": "Phillips", "given": "Robert B." }, "role": "member" }, { "email": "melowitz@caltech.edu", "id": "Elowitz-M-B", "name": { "family": "Elowitz", "given": "Michael B." }, "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2009-08-03", "gradofc_approval_date": "2010-02-01", "review_status": "approved", "option_major": { "items": [ "bioeng" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2010", "author_list": "Choi, Harry Ming Tak", "advisor_list": "Pierce, Niles A.", "comittee_list": "Fraser, Scott E.; Phillips, Robert B.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/5801", "eprint_id": 5801, "rev_number": 27, "documents": [ { "id": "/id/document/10054", "doc_id": 10054, "rev_number": 5, "files": [ { "id": "/id/file/65327", "fileid": 65327, "datasetid": "document", "objectid": 10054, "filename": "thesis.pdf", "mime_type": "application/pdf", "filesize": 5783929, "mtime": "2012-12-26 03:25:39", "url": "/5801/1/thesis.pdf" } ], "eprint_id": 5801, "pos": 1, "mime_type": "application/pdf", "format": "application/pdf", "language": "en", "security": "public", "license": "other", "main": "thesis.pdf", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", 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"http://eprints.org/relation/isVersionOf", "uri": "/id/document/10054" }, { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/10054" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/10054" } ] } } ], "eprint_status": "archive", "userid": 1502, "dir": "disk0/00/00/58/01", "datestamp": "2010-05-25 16:07:33", "lastmod": "2023-11-08 00:43:32", "status_changed": "2010-05-25 16:07:33", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "zadeh@caltech.edu", "id": "Zadeh-Joseph-N", "name": { "family": "Zadeh", "given": "Joseph N." }, "show_email": "NO" } ] }, "title": "Algorithms for Nucleic Acid Sequence Design", "ispublished": "unpub", "full_text_status": "public", "keywords": "RNA, DNA, secondary structure, sequence design, partition function", "abstract": "Motivated by a growing field of research focused on programming function into biomolecules, we seek to decrease the cost of high-quality rational nucleic acid sequence design while increasing its versatility and availability. We begin by describing an algorithm for designing the sequence of one or more interacting nucleic acid strands intended to adopt a target secondary structure at equilibrium. Using ensemble defect optimization, we seek to minimize the average number of incorrectly paired nucleotides at equilibrium, calculated over the entire ensemble of unpseudoknotted secondary structures. Empirically, the algorithm exhibits asymptotic optimality and costs 4/3 the time of a single objective function evaluation for large structures. We then extend this algorithm to design multi-state systems with an arbitrary number of linked targets and demonstrate its efficacy on systems invented by molecular engineers. To improve the ease of use and availability of nucleic acid analysis and design tools, we present NUPACK, a web application already in wide use that allows the international research community to share a high-performance compute cluster for the analysis and design of systems of interacting nucleic acids. ", "date": "2010", "date_type": "degree", "id_number": "CaltechTHESIS:05112010-205335518", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:05112010-205335518", "related_url": { "items": [ { "description": "NUPACK web application", "type": "related", "url": "http://www.nupack.org" } ] }, "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Gayle Hammer", "deposited_by": "Joseph Zadeh", "deposited_on": "2010-05-25 16:07:33", "doi": "10.7907/20DG-NF39", "divisions": { "items": [ "div_eng" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "winfree@caltech.edu", "id": "Winfree-E", "name": { "family": "Winfree", "given": "Erik" }, "role": "chair" }, { "email": "pwkr@dna.caltech.edu", "id": "Rothemund-P-W-K", "name": { "family": "Rothemund", "given": "Paul W. K." }, "role": "member" }, { "email": "steve@mayo.caltech.edu", "id": "Mayo-S-L", "name": { "family": "Mayo", "given": "Stephen L." }, "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2009-12-08", "gradofc_approval_date": "2010-05-24", "review_status": "approved", "option_major": { "items": [ "bioeng" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2010", "author_list": "Zadeh, Joseph N.", "advisor_list": "Pierce, Niles A.", "comittee_list": "Winfree, Erik; Rothemund, Paul W. 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design and analysis of nucleic acid secondary structures, we seek to develop efficient and scalable algorithms for the analysis of secondary structure kinetics. Secondary structure kinetics are modeled by a first-order master equation, but the number of secondary structures for a sequence grows exponentially with the length of the sequence, meaning that for systems of interest, we cannot write down the rate matrix, much less solve the master equation. To address these difficulties, we develop a method to construct macrostate maps of nucleic acid free energy landscapes based on simulating the continuous-time Markov chain associated with the microstate master equation. The method relies on the careful combination of several elements: a novel procedure to explicitly identify transitions between macrostates in the simulation, a goodness-of-clustering test specific to secondary structures, an algorithm to find the centroid secondary structure for each macrostate, a method to compute macrostate partition functions from short simulations, and a framework for computing transition rates with confidence intervals. We use this method to study several experimental systems from our laboratory with system sizes in the hundreds of nucleotides, and develop a model problem, the d-cube, for which we can control all of the relevant parameters and analyze our method's error behavior. Our results and analysis suggest that this method will be useful not only in the analysis and design of nucleic acid mechanical devices, but also in wider applications of molecular simulation and simulation-based model reduction.", "date": "2009", "date_type": "degree", "id_number": "CaltechETD:etd-12312008-153810", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechETD:etd-12312008-153810", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Restricted to Caltech community only until 31 May 2020.", "deposited_by": "Imported from ETD-db", "deposited_on": "2009-05-13", "doi": "10.7907/VJX1-6376", "divisions": { "items": [ "div_eng" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "chair" }, { "email": "dim@caltech.edu", "id": "Meiron-D-I", "name": { "family": "Meiron", "given": "Daniel I." }, "role": "member" }, { "email": "owhadi@caltech.edu", "id": "Owhadi-H", "name": { "family": "Owhadi", "given": "Houman" }, "role": "member" }, { "email": "jmarsden@caltech.edu", "id": "Marsden-J-E", "name": { "family": "Marsden", "given": "Jerrold E." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_submitted_date": "2008-12-31", "thesis_defense_date": "2008-06-09", "thesis_approved_date": "2009-05-13", "gradofc_approval_date": "2009-05-12", "review_status": "approved", "option_major": { "items": [ "appliedmath" ] }, "copyright_statement": "I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive\nlicense to archive and make accessible, under the conditions specified below,\nmy thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2009", "author_list": "Othmer, Jonathan Andrew", "advisor_list": "Pierce, Niles A.", "comittee_list": "Pierce, Niles A.; Meiron, Daniel I.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/2032", "eprint_id": 2032, "rev_number": 12, "documents": [ { "id": "/id/document/3637", "doc_id": 3637, "rev_number": 3, "files": [ { "id": "/id/file/21408", "fileid": 21408, "datasetid": "document", "objectid": 3637, "filename": "thesis_electronic.pdf", "mime_type": "application/pdf", "filesize": 2659724, "mtime": "2012-12-26 02:46:03", "url": "/2032/1/thesis_electronic.pdf" } ], "eprint_id": 2032, "pos": 1, "mime_type": "application/pdf", "format": "application/pdf", "language": "en", "security": "public", "license": "other", "main": "thesis_electronic.pdf", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "content": "final", "relation": { "items": [ { "type": "http://eprints.org/relation/hasVolatileVersion", "uri": "/id/document/20305" }, { "type": "http://eprints.org/relation/haspreviewThumbnailVersion", "uri": "/id/document/20305" }, { "type": "http://eprints.org/relation/hasVersion", "uri": "/id/document/20305" } ] } }, { "id": "/id/document/15716", "doc_id": 15716, "rev_number": 3, "files": [ { "id": "/id/file/202382", "fileid": 202382, "datasetid": "document", "objectid": 15716, "filename": "Bois_Justin_S_2007.PDF", "mime_type": "application/pdf", "filesize": 86090, "mtime": "2016-08-22 21:15:29", "url": "/2032/2/Bois_Justin_S_2007.PDF" } ], "eprint_id": 2032, "pos": 2, "format": "application/pdf", "language": "en", "security": "internal", "license": "other", "main": "Bois_Justin_S_2007.PDF", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "content": "approvalform" }, { "id": "/id/document/20305", "doc_id": 20305, "rev_number": 2, "files": [ { "id": "/id/file/21406", "fileid": 21406, "datasetid": "document", "objectid": 20305, "filename": "preview.png", "mime_type": "image/png", "hash": "24c2d9347fc96fd38154062c62ca3ba1", "hash_type": "MD5", "filesize": 12463, "mtime": "2012-12-26 02:46:03", "url": "/2032/3/preview.png" } ], "eprint_id": 2032, "pos": 3, "placement": 3, "mime_type": "image/png", "format": "image/png", "language": "en", "security": "public", "license": "other", "main": "preview.png", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/3637" }, { "type": "http://eprints.org/relation/ispreviewThumbnailVersionOf", "uri": "/id/document/3637" }, { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/3637" } ] } }, { "id": "/id/document/121923", "doc_id": 121923, "rev_number": 1, "files": [ { "id": "/id/file/367317", "fileid": 367317, "datasetid": "document", "objectid": 121923, "filename": "indexcodes.txt", "mime_type": "text/plain", "hash": "d82892cb54f41692eca14227359af261", "hash_type": "MD5", "filesize": 25655, "mtime": "2021-06-23 21:41:04", "url": "/2032/4/indexcodes.txt" } ], "eprint_id": 2032, "pos": 4, "placement": 4, "mime_type": "text/plain", "format": "other", "format_desc": "Generate index codes conversion from application/pdf to indexcodes", "language": "en", "security": "public", "license": "other", "main": "indexcodes.txt", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/3637" }, { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/3637" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/3637" } ] } } ], "eprint_status": "archive", "userid": 2, "dir": "disk0/00/00/20/32", "datestamp": "2007-05-31", "lastmod": "2023-11-08 00:43:32", "status_changed": "2009-09-25 02:24:04", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "bois@caltech.edu", "id": "Bois-Justin-S", "name": { "family": "Bois", "given": "Justin S." }, "orcid": "0000-0001-7137-8746", "show_email": "YES" } ] }, "title": "Analysis of Interacting Nucleic Acids in Dilute Solutions", "ispublished": "unpub", "full_text_status": "public", "keywords": "base pair; convexity; dilute solution; distinguishability; DNA; duality; equilibrium; folding; multiple strands; nucleic acid; NUPACK; partition function; redundancy; RNA; secondary structure; symmetry; thermodynamics", "abstract": "Motivated by the growing demand for analysis tools for diverse natural and engineered DNA and RNA systems, we develop a general theory and set of computational algorithms to perform thermodynamic analysis of dilute reactive solutions and then apply these techniques to interacting nucleic acids. The theory correctly accounts for the effects of indistinguishability in partition function calculations for complexes of interacting strands. With partition functions in hand, the unique complex concentrations corresponding to thermodynamic equilibrium are obtained by solving a convex programming problem. Partition function and concentration information can then be used to calculate equilibrium base-pairing observables corresponding to experimentally measurable properties. The underlying physics and mathematical formulation of these problems lead to an interesting blend of approaches, including ideas from graph theory, group theory, dynamic programming, combinatorics, convex optimization, and Lagrange duality.
\r\n\r\nTo make these analysis tools available to researchers worldwide, we present NUPACK, a web-based software suite for thermodynamic analysis of nucleic acids. Its efficacy is demonstrated in example calculations and the results are shown to be in agreement with experiment.
\r\n\r\nFinally, the thermodynamic properties of a DNA-based triggered self-assembly device [1] are analyzed using NUPACK and extensions of its tools. The computational results complement experimental studies, exposing novel properties about the system and dictating further research.
", "date": "2007", "date_type": "degree", "id_number": "CaltechETD:etd-05242007-211446", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechETD:etd-05242007-211446", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "deposited_by": "Imported from ETD-db", "deposited_on": "2007-05-31", "doi": "10.7907/2JCJ-SX75", "divisions": { "items": [ "div_chem" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "zgw@caltech.edu", "id": "Wang-Zhen-Gang", "name": { "family": "Wang", "given": "Zhen-Gang" }, "role": "chair" }, { "email": "smolke@cheme.caltech.edu", "id": "Smolke-C-D", "name": { "family": "Smolke", "given": "Christina D." }, "role": "member" }, { "email": "winfree@caltech.edu", "id": "Winfree-E", "name": { "family": "Winfree", "given": "Erik" }, "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_submitted_date": "2007-05-24", "thesis_defense_date": "2007-04-30", "thesis_approved_date": "2007-05-31", "gradofc_approval_date": "2007-05-30", "review_status": "approved", "option_major": { "items": [ "chemeng" ] }, "copyright_statement": "I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive\nlicense to archive and make accessible, under the conditions specified below,\nmy thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. 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"title": "Analysis, Design, and Construction of Nucleic Acid Devices", "ispublished": "unpub", "full_text_status": "public", "keywords": "Design; DNA; HCR; Hybridization Chain Reaction; Nucleic acids; Pseudoknots; RNA", "abstract": "Nucleic acids present great promise as building blocks for nanoscale devices. To achieve this potential, methods for the analysis and design of DNA and RNA need to be improved. In this thesis, traditional algorithms for analyzing nucleic acids at equilibrium are extended to handle a class of pseudoknots, with examples provided relevant to biologists and bioengineers. With these analytical tools in hand, nucleic acid sequences are designed to maximize the equilibrium probability of a desired fold. Upon analysis, it is concluded that both affinity and specificity are important when choosing a sequence; this conclusion holds for a wide range of target structures and is robust to random perturbations to the energy model. Applying the intuition gained from these studies, a process called hybridization chain reaction (HCR) is invented, and sequences are chosen that experimentally verify this phenomenon. In HCR, a small number of DNA or RNA molecules trigger a system wide configurational change, allowing the amplification and detection of specific, nucleic acid sequences. As an extension, HCR is combined with a pre-existing aptamer domain to successfully construct an ATP sensor, and the groundwork is laid for the future development of sensors for other small molecules. In addition, recent studies on multi-stranded algorithms and improvements to HCR are included in the appendices. Not only will these advancements increase our understanding of biological RNAs, but they will also provide valuable tools for the future development of nucleic acid nanotechnologies.", "date": "2005", "date_type": "degree", "id_number": "CaltechETD:etd-05242005-133116", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechETD:etd-05242005-133116", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "suggestions": "Robert Dirks tragically lost his life in the Metro-North train crash in New York on February 3, 2015. Robert wrote the first lines of code for NUPACK and made indelible contributions to its algorithms. Brilliant, creative, generous, humble, fun-loving, and kind, Robert was a model scientist, collaborator, and friend. Donations in Robert's honor may be made to the Dirks Prize Fund. (http://www.nupack.org/home/contact)", "deposited_by": "Imported from ETD-db", "deposited_on": "2005-05-26", "doi": "10.7907/VZ43-FM66", "divisions": { "items": [ "div_chem" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." } } ] }, "thesis_committee": { "items": [ { "email": "steve@mayo.caltech.edu", "id": "Mayo-S-L", "name": { "family": "Mayo", "given": "Stephen L." }, "role": "chair" }, { "email": "nslewis@its.caltech.edu", "id": "Lewis-N-S", "name": { "family": "Lewis", "given": "Nathan Saul" }, "role": "member" }, { "email": "niles@caltech.edu", "id": "Pierce-N-A", "name": { "family": "Pierce", "given": "Niles A." }, "role": "member" }, { "email": "wag@caltech.edu", "id": "Goddard-W-A-III", "name": { "family": "Goddard", "given": "William A., III" }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_submitted_date": "2005-05-24", "thesis_defense_date": "2005-05-11", "thesis_approved_date": "2005-05-26", "review_status": "approved", "option_major": { "items": [ "chemistry" ] }, "copyright_statement": "I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive\nlicense to archive and make accessible, under the conditions specified below,\nmy thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2005", "author_list": "Dirks, Robert Michael", "advisor_list": "Pierce, Niles A.", "comittee_list": "Mayo, Stephen L.; Lewis, Nathan Saul; et el." } ]