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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Article
  • Peer Reviewed
UC San Diego Previously Published Works (2023)
Combinations of biological macromolecules can provide researchers with precise control and unique methods for regulating, studying, and manipulating cellular processes. For instance, combining the unmatched encodability afforded by nucleic acids with the diverse functionality of proteins has transformed our approach to solving several problems in chemical biology. Despite these benefits, there remains a need for new methods to site-specifically generate conjugates between different classes of biomolecules. Here we present a fully enzymatic strategy for combining nucleic acids and proteins using SNAP-tag and RNA-TAG (transglycosylation at guanosine) technologies via a bifunctional preQ1-benzylguanine small molecule probe. We demonstrate the robust ability of this technology to assemble site-specific SNAP-tag - RNA conjugates with RNAs of varying length and use our conjugation strategy to recruit an endonuclease to an RNA of interest for targeted degradation. We foresee that combining SNAP-tag and RNA-TAG will facilitate researchers to predictably engineer novel macromolecular complexes.
    Cover page: RNA-TAG Mediated Protein-RNA Conjugation.
    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2021)

    The importance of nucleic acids has expended past their role in encoding life and into a space of scientific utility and discovery. Developing new, robust methods to study and manipulate them in their natural environments and harness their programmability for fundamental research tools and biomedical applications has become a critical goal for researchers. This work presents an enzymatic tool for functional modification of single stranded DNA oligonucleotides as well a strategy to assemble functional nucleic acid protein conjugates.

    Due to its greater stability and ease of synthesis, DNA, rather than RNA, is the nucleic acid of choice for many functional applications. Often, these applications require the DNA oligonucleotide to be modified in some way with an affinity tag or reporter. There are a limited number of methods to label DNA oligonucleotides, all of which have their shortcomings, including being cost prohibitive, inefficient, or a limited small molecule scope. In this dissertation I develop an enzymatic single stranded DNA modification method, expanding on the powerful RNA transglycosylation at guanine (RNA-TAG) modification tool developed in our lab. DNA-TAG offers researchers with an unprecedented tool to generate modified oligonucleotides in their lab. This work describes the rational and iterative design of DNA-TAG and demonstrates its utility through fluorescent northern blot and RNA fluorescent in situ hybridization (FISH).

    Combining the programmability of nucleic acids with the functions of proteins has the potential to enable powerful assemblies with unique and critical functions, both natural and synthetic. Developing novel strategies to bring these classes of biomolecules together in a robust and specific manner continues to facilitate new applications. Here I present a fully enzymatic method to covalently assemble a functional RNA-Protein conjugate using a novel bifunctional small molecule probe and demonstrate he utility of the system by recruitment of an endonuclease to an RNA of interest to induce degradation.

    With the herein presented advancements, we expand the application space of nucleic acid modification. Nucleic acid transglycosylation at guanine (NA-TAG) technologies provide researchers with a single tool capable of modifying nearly any single stranded nucleic acid substrate with a small molecule of their choice for downstream applications.

      Cover page: Enzymatic Tools for Enabling Nucleic Acid Driven Technologies
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2023)
      Bacterial tRNA guanine transglycosylases (TGTs) catalyze the exchange of guanine for the 7-deazaguanine queuine precursor, prequeuosine1 (preQ1). While the native nucleic acid substrate for bacterial TGTs is the anticodon loop of queuine-cognate tRNAs, the minimum recognition sequence for the enzyme is a structured hairpin containing the target G nucleobase in a "UGU" loop motif. Previous work has established an RNA modification system, RNA-TAG, in which Escherichia coli TGT exchanges the target G on an RNA of interest for chemically modified preQ1 substrates linked to a small-molecule reporter such as biotin or a fluorophore. While extending the substrate scope of RNA transglycosylases to include DNA would enable numerous applications, it has been previously reported that TGT is incapable of modifying native DNA. Here, we demonstrate that TGT can in fact recognize and label specific DNA substrates. Through iterative testing of rationally mutated DNA hairpin sequences, we determined the minimal sequence requirements for transglycosylation of unmodified DNA by E. coli TGT. Controlling steric constraint in the DNA hairpin dramatically affects labeling efficiency, and, when optimized, can lead to near-quantitative site-specific modification. We demonstrate the utility of our newly developed DNA-TAG system by rapidly synthesizing probes for fluorescent Northern blotting of spliceosomal U6 RNA and RNA FISH visualization of the long noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The ease and convenience of the DNA-TAG system will provide researchers with a tool for accessing a wide variety of versatile and affordable modified DNA substrates.
        Cover page: Site-Specific Covalent Labeling of DNA Substrates by an RNA Transglycosylase
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2022)
        Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the cross-linker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting gene editing. Visible light irradiation cleaved the cross-linker and restored gene editing with high spatiotemporal resolution. Design of two photocleavable linkers responsive to different wavelengths of light allowed multiplexed photoactivation of gene editing in mammalian cells. This photoactivated CRISPR-Cas9 gene editing platform benefits from undetectable background activity, provides a choice of activation wavelengths, and has multiplexing capabilities.
          Cover page: Site-Specific and Enzymatic Cross-Linking of sgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2020)
          Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2020)
            Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
              Cover page: Proteomics-based screening of the endothelial heparan sulfate interactome reveals that C-type lectin 14a (CLEC14A) is a heparin-binding protein
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