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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2020)

RNA is one of the most important biomacromolecules in the living systems, manipulating a highly complexed collection of functions which are critical to the regulation of numerous cellular processes. In the past few years, our lab has developed a versatile and powerful RNA modifying technique, named RNA-TAG. The RNA-TAG technique utilizes a bacterial tRNA guanine transglycosylase (TGT) to exchange a guanine nucleobase within a specific 17-nucleotide motif (Tag) for synthetic pre-queuosine1 (preQ1) derivatives. By inserting this Tag sequence into an RNA of interest, we can covalently and site-specifically conjugate functional small-molecules onto any RNA of interest. I applied our RNA-TAG technique to manipulate the functions of different types of cellular RNA, for example, mRNA for translation regulation and the singe guide RNA (sgRNA) for CRISPR/Cas9 gene editing.To interfere with the translation process of mature mRNA, I systematically conjugated three bulky visible-light photocleavable caging groups along the 5’ untranslated region (5’-UTR) of an mRNA, severely reducing its translation activity. Upon visible-light photocleavage of these caging groups, mRNA translation was resorted. To expand our technology, we further designed two photo-sensitive caging groups which can be sequentially cleaved by two wavelength of lights (405 nm and 488 nm). In this manner, a sequential/multiplexed photo-activation of two mRNAs within the same cells was achieved. Based on our RNA-TAG technique, I developed a novel technique named RNA-CLAMP, which can covalently and site-specifically crosslink (‘clamp’) two internal stem-loops within a RNA of interest. By designing small-molecule substrates which contain a cleavable linker and two preQ1 moieties, I achieved accurate ‘clamping’ and rapid release of the target RNA, which significantly alter its secondary/ternary structure, resulting in gain/loss of functions. I applied the RNA-CLAMP technique to the sgRNA of the CRISPR/Cas9 gene editing system. Upon ‘clamping’ of two internal loops (the tetra-loop and stem-loop2) of the sgRNA, the Cas9 gene editing system was completely deactivated. However, the cleavage of the crosslinker with an external stimulus, for example, light irradiation, released the sgRNA and fully activated gene editing in live mammalian cells. To the best of knowledge, this CRISPR/Cas9 gene editing photo-regulation technology offers the best dynamic range, flexibility, multiplexing capability and accessibility to date. We believe with further development, the RNA-CLAMP technique will have tremendous applications in manipulating other types of RNAs, such as ribozymes, microRNAs, and long non-coding RNAs. Such techniques will serve as powerful tools in studying complex cellular networks as well as developing novel therapeutics applications.

    Cover page: Control the Function of RNA by Site-Specific Bioconjugation
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2022)
    Bioorthogonal cycloaddition reactions between tetrazines and strained dienophiles are widely used for protein, lipid and glycan labelling because of their extremely rapid kinetics. However, controlling this chemistry in the presence of living mammalian cells with a high degree of spatial and temporal precision remains a challenge. Here we demonstrate a versatile approach to light-activated formation of tetrazines from photocaged dihydrotetrazines. Photouncaging, followed by spontaneous transformation to reactive tetrazine, enables live-cell spatiotemporal control of rapid bioorthogonal cycloaddition with dienophiles such as trans-cyclooctenes. Photocaged dihydrotetrazines are stable in conditions that normally degrade tetrazines, enabling efficient early-stage incorporation of bioorthogonal handles into biomolecules such as peptides. Photocaged dihydrotetrazines allow the use of non-toxic light to trigger tetrazine ligations on living mammalian cells. By tagging reactive phospholipids with fluorophores, we demonstrate modification of HeLa cell membranes with single-cell spatial resolution. Finally, we show that photo-triggered therapy is possible by coupling tetrazine photoactivation with strategies that release prodrugs in response to tetrazine ligation.
      Cover page: Light-activated tetrazines enable precision live-cell bioorthogonal chemistry
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2020)
      All living organisms synthesize phospholipids as the primary constituent of their cell membranes. Enzymatic synthesis of diacylphospholipids requires preexisting membrane-embedded enzymes. This limitation has led to models of early life in which the first cells used simpler types of membrane building blocks and has hampered integration of phospholipid synthesis into artificial cells. Here we demonstrate an enzyme-free synthesis of natural diacylphospholipids by transacylation in water, which is enabled by a combination of ion pairing and self-assembly between lysophospholipids and acyl donors. A variety of membrane-forming cellular phospholipids have been obtained in high yields. Membrane formation takes place in water from natural alkaline sources such as soda lakes and hydrothermal oceanic vents. When formed vesicles are transferred to more acidic solutions, electrochemical proton gradients are spontaneously established and maintained. This high-yielding non-enzymatic synthesis of natural phospholipids in water opens up new routes for lipid synthesis in artificial cells and sheds light on the origin and evolution of cellular membranes.
        Cover page: Enzyme-free synthesis of natural phospholipids in water
        • 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 (2024)
          Mutations in the Cockayne Syndrome group B (CSB) gene cause cancer in mice, but premature aging and severe neurodevelopmental defects in humans. CSB, a member of the SWI/SNF family of chromatin remodelers, plays diverse roles in regulating gene expression and transcription-coupled nucleotide excision repair (TC-NER); however, these functions do not explain the distinct phenotypic differences observed between CSB-deficient mice and humans. During investigating Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism that involves elongating RNA polymerase II (RNAPII) undergoing transient pauses at internal T-runs where CSB is required to propel RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled with G-rich sequences upstream, exacerbates genome instability by promoting R-loop formation. These R-loop prone motifs are notably abundant in relatively long genes related to neuronal functions in the human genome, but less prevalent in the mouse genome. These findings provide mechanistic insights into differential impacts of CSB deficiency on mice versus humans and suggest that the manifestation of the Cockayne Syndrome phenotype in humans results from the progressive evolution of mammalian genomes.
            Cover page: Cockayne Syndrome Linked to Elevated R-Loops Induced by Stalled RNA Polymerase II during Transcription Elongation
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2020)
            Parkinson's disease is characterized by loss of dopamine neurons in the substantia nigra1. Similar to other major neurodegenerative disorders, there are no disease-modifying treatments for Parkinson's disease. While most treatment strategies aim to prevent neuronal loss or protect vulnerable neuronal circuits, a potential alternative is to replace lost neurons to reconstruct disrupted circuits2. Here we report an efficient one-step conversion of isolated mouse and human astrocytes to functional neurons by depleting the RNA-binding protein PTB (also known as PTBP1). Applying this approach to the mouse brain, we demonstrate progressive conversion of astrocytes to new neurons that innervate into and repopulate endogenous neural circuits. Astrocytes from different brain regions are converted to different neuronal subtypes. Using a chemically induced model of Parkinson's disease in mouse, we show conversion of midbrain astrocytes to dopaminergic neurons, which provide axons to reconstruct the nigrostriatal circuit. Notably, re-innervation of striatum is accompanied by restoration of dopamine levels and rescue of motor deficits. A similar reversal of disease phenotype is also accomplished by converting astrocytes to neurons using antisense oligonucleotides to transiently suppress PTB. These findings identify a potentially powerful and clinically feasible approach to treating neurodegeneration by replacing lost neurons.
              Cover page: Reversing a model of Parkinson’s disease with in situ converted nigral neurons
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