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  1. Article ; Online: Reply to: On gene silencing by the X10-23 DNAzyme.

    Spitale, Robert C / Chaput, John C

    Nature chemistry

    2022  Volume 14, Issue 8, Page(s) 859–861

    MeSH term(s) DNA, Catalytic/genetics ; Gene Silencing ; RNA, Messenger/genetics
    Chemical Substances DNA, Catalytic ; RNA, Messenger
    Language English
    Publishing date 2022-07-18
    Publishing country England
    Document type Letter ; Research Support, Non-U.S. Gov't ; Comment
    ZDB-ID 2464596-5
    ISSN 1755-4349 ; 1755-4330
    ISSN (online) 1755-4349
    ISSN 1755-4330
    DOI 10.1038/s41557-022-00983-4
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Probing the dynamic RNA structurome and its functions.

    Spitale, Robert C / Incarnato, Danny

    Nature reviews. Genetics

    2022  Volume 24, Issue 3, Page(s) 178–196

    Abstract: RNA is a key regulator of almost every cellular process, and the structures adopted by RNA molecules are thought to be central to their functions. The recent fast-paced evolution of high-throughput sequencing-based RNA structure mapping methods has ... ...

    Abstract RNA is a key regulator of almost every cellular process, and the structures adopted by RNA molecules are thought to be central to their functions. The recent fast-paced evolution of high-throughput sequencing-based RNA structure mapping methods has enabled the rapid in vivo structural interrogation of entire cellular transcriptomes. Collectively, these studies are shedding new light on the long underestimated complexity of the structural organization of the transcriptome - the RNA structurome. Moreover, recent analyses are challenging the view that the RNA structurome is a static entity by revealing how RNA molecules establish intricate networks of alternative intramolecular and intermolecular interactions and that these ensembles of RNA structures are dynamically regulated to finely tune RNA functions in living cells. This new understanding of how RNA can shape cell phenotypes has important implications for the development of RNA-targeted therapeutic strategies.
    MeSH term(s) RNA/genetics ; Nucleic Acid Conformation ; Transcriptome ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, RNA/methods
    Chemical Substances RNA (63231-63-0)
    Language English
    Publishing date 2022-11-08
    Publishing country England
    Document type Journal Article ; Review ; Research Support, Non-U.S. Gov't
    ZDB-ID 2035157-4
    ISSN 1471-0064 ; 1471-0056
    ISSN (online) 1471-0064
    ISSN 1471-0056
    DOI 10.1038/s41576-022-00546-w
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Using 5'

    Garfio, Chely M / Gupta, Mrityunjay / Spitale, Robert C

    Current protocols

    2023  Volume 3, Issue 7, Page(s) e830

    Abstract: RNA molecules perform numerous cellular functions necessary for cell viability, some of which can depend on the RNA's structure. Therefore, it is important to study RNA structure and RNA folding to better understand the molecular basis of these functions. ...

    Abstract RNA molecules perform numerous cellular functions necessary for cell viability, some of which can depend on the RNA's structure. Therefore, it is important to study RNA structure and RNA folding to better understand the molecular basis of these functions. RNA chemical mapping strategies to elucidate RNA structural changes involve using chemical reagents that form adducts or cleave RNA. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) measures RNA flexibility by modification of the 2' hydroxyl groups of flexible nucleotides. These RNA adducts can be detected using
    MeSH term(s) RNA/genetics ; RNA/chemistry ; Nucleic Acid Conformation ; Reverse Transcription ; RNA Folding ; Hydroxyl Radical/chemistry
    Chemical Substances RNA (63231-63-0) ; Hydroxyl Radical (3352-57-6)
    Language English
    Publishing date 2023-07-20
    Publishing country United States
    Document type Journal Article
    ISSN 2691-1299
    ISSN (online) 2691-1299
    DOI 10.1002/cpz1.830
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  4. Article ; Online: Overview of Chemical Methods to Probe RNA Structure with Radionucleotides.

    Gupta, Mrityunjay / Garfio, Chely M / Spitale, Robert C

    Current protocols

    2023  Volume 3, Issue 5, Page(s) e781

    Abstract: Structural features of RNA play an important role in its capability to perform various functions in biological systems. To probe structural features, chemical probes are used to conjugate or cleave RNA at solvent-accessible sites, differentiating between ...

    Abstract Structural features of RNA play an important role in its capability to perform various functions in biological systems. To probe structural features, chemical probes are used to conjugate or cleave RNA at solvent-accessible sites, differentiating between flexible and constrained regions. These conjugates or cleaved products are then detected using reverse transcription (RT), where enzymatic RNA-dependent DNA primer extension is abruptly halted at the conjugation site or cleavage site. Here, we provide an overview of methods to probe RNA structure in vitro using radioactively labeled DNA primers, which provide a highly sensitive method to visualize RT stop sites with gel electrophoresis. © 2023 Wiley Periodicals LLC.
    MeSH term(s) RNA/genetics ; RNA/chemistry ; DNA/analysis ; Reverse Transcription ; DNA Primers/chemistry
    Chemical Substances RNA (63231-63-0) ; DNA (9007-49-2) ; DNA Primers
    Language English
    Publishing date 2023-02-20
    Publishing country United States
    Document type Journal Article
    ISSN 2691-1299
    ISSN (online) 2691-1299
    DOI 10.1002/cpz1.781
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  5. Article ; Online: mRNAs encoding neurodevelopmental regulators have equal N6-methyladenosine stoichiometry in Drosophila neuroblasts and neurons.

    Sami, Josephine D / Spitale, Robert C / Cleary, Michael D

    Neural development

    2022  Volume 17, Issue 1, Page(s) 9

    Abstract: ... ...

    Abstract N
    MeSH term(s) Adenosine/analogs & derivatives ; Adenosine/genetics ; Adenosine/metabolism ; Animals ; Drosophila ; Drosophila melanogaster ; Methyltransferases/genetics ; Methyltransferases/metabolism ; Neurons/metabolism ; RNA, Messenger/metabolism
    Chemical Substances RNA, Messenger ; N-methyladenosine (CLE6G00625) ; Methyltransferases (EC 2.1.1.-) ; Adenosine (K72T3FS567)
    Language English
    Publishing date 2022-10-15
    Publishing country England
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural
    ZDB-ID 2254847-6
    ISSN 1749-8104 ; 1749-8104
    ISSN (online) 1749-8104
    ISSN 1749-8104
    DOI 10.1186/s13064-022-00166-4
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  6. Article ; Online: Assaying RNA solvent accessibility in living cells with LASER.

    Feng, Chao / Spitale, Robert C

    Methods in enzymology

    2020  Volume 641, Page(s) 401–411

    Abstract: RNA molecules perform a wide variety of functions to control many cellular pathways. Critical to these roles is the ability of an RNA to fold into complex three-dimensional structures. As part of this folding, unique elements of RNA structure become more ...

    Abstract RNA molecules perform a wide variety of functions to control many cellular pathways. Critical to these roles is the ability of an RNA to fold into complex three-dimensional structures. As part of this folding, unique elements of RNA structure become more exposed or hidden to solvent and these properties are important to understand an RNAs final fold. Characterizing solvent accessibility can enable researchers to better understand the overall fold of an RNA and how it interacts with trans acting factors, such as proteins. Herein we describe the methods toward using light activated structural evaluation of RNA, or LASER, which measures purine nucleobase solvent accessibility. To date, LASER has been used inside and outside of cells to measure RNA solvent accessibility and RNA interactions with proteins.
    MeSH term(s) Lasers ; Light ; Nucleic Acid Conformation ; RNA ; Solvents
    Chemical Substances Solvents ; RNA (63231-63-0)
    Language English
    Publishing date 2020-06-15
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ISSN 1557-7988
    ISSN (online) 1557-7988
    DOI 10.1016/bs.mie.2020.04.046
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  7. Article ; Online: Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides.

    Gupta, Mrityunjay / Levine, Samantha R / Spitale, Robert C

    Accounts of chemical research

    2022  Volume 55, Issue 18, Page(s) 2647–2659

    Abstract: ConspectusThe discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. ... ...

    Abstract ConspectusThe discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling.To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels-Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological systems.
    MeSH term(s) Nucleosides ; RNA/chemistry ; Transferases ; Uracil ; Uridine Monophosphate
    Chemical Substances Nucleosides ; Uracil (56HH86ZVCT) ; RNA (63231-63-0) ; Uridine Monophosphate (E2OU15WN0N) ; Transferases (EC 2.-)
    Language English
    Publishing date 2022-09-08
    Publishing country United States
    Document type Journal Article
    ZDB-ID 1483291-4
    ISSN 1520-4898 ; 0001-4842
    ISSN (online) 1520-4898
    ISSN 0001-4842
    DOI 10.1021/acs.accounts.2c00347
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  8. Article ; Online: Cycloaddition enabled mutational profiling of 5-vinyluridine in RNA.

    Gupta, Mrityunjay / Wang, Jingtian / Garfio, Chely M / Vandewalle, Abigail / Spitale, Robert C

    Chemical communications (Cambridge, England)

    2023  Volume 59, Issue 22, Page(s) 3257–3260

    Abstract: We report the detection of 5-vinyluridine (5-VUrd) in RNA at single nucleotide ... ...

    Abstract We report the detection of 5-vinyluridine (5-VUrd) in RNA at single nucleotide resolution
    MeSH term(s) RNA/genetics ; Cycloaddition Reaction ; Nucleotides
    Chemical Substances RNA (63231-63-0) ; Nucleotides
    Language English
    Publishing date 2023-03-14
    Publishing country England
    Document type Journal Article
    ZDB-ID 1472881-3
    ISSN 1364-548X ; 1359-7345 ; 0009-241X
    ISSN (online) 1364-548X
    ISSN 1359-7345 ; 0009-241X
    DOI 10.1039/d3cc00043e
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  9. Article ; Online: Repurposing a DNA-Repair Enzyme for Targeted Protein Degradation.

    Fredlender, Callie / Levine, Samantha / Zimak, Jan / Spitale, Robert C

    Chembiochem : a European journal of chemical biology

    2022  Volume 23, Issue 19, Page(s) e202200053

    Abstract: Herein we present the exploration of the utility of DNA demethylase enzymes for targeted protein degradation. Novel benzylguanine substrates are characterized for their ability to control protein degradation in cells. Our data demonstrate the utility of ... ...

    Abstract Herein we present the exploration of the utility of DNA demethylase enzymes for targeted protein degradation. Novel benzylguanine substrates are characterized for their ability to control protein degradation in cells. Our data demonstrate the utility of this approach to degrade fusion proteins in different localizations within living cells.
    MeSH term(s) DNA Repair Enzymes ; Proteolysis ; Recombinant Fusion Proteins
    Chemical Substances Recombinant Fusion Proteins ; DNA Repair Enzymes (EC 6.5.1.-)
    Language English
    Publishing date 2022-06-24
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 2020469-3
    ISSN 1439-7633 ; 1439-4227
    ISSN (online) 1439-7633
    ISSN 1439-4227
    DOI 10.1002/cbic.202200053
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  10. Article ; Online: A Fluorescent Reverse-Transcription Assay to Detect Chemical Adducts on RNA.

    Falco, Natalie / Garfio, Chely M / Spitalny, Leslie / Spitale, Robert C

    Biochemistry

    2022  Volume 61, Issue 16, Page(s) 1665–1668

    Abstract: Herein, we detail a novel reverse-transcription (RT) assay to directly detect chemical adducts on RNA. We optimize a fluorescence quenching assay to detect RT polymerization and employ our approach to detect ... ...

    Abstract Herein, we detail a novel reverse-transcription (RT) assay to directly detect chemical adducts on RNA. We optimize a fluorescence quenching assay to detect RT polymerization and employ our approach to detect N
    MeSH term(s) Alkylation ; Inosine ; RNA/chemistry ; Reverse Transcription
    Chemical Substances Inosine (5A614L51CT) ; RNA (63231-63-0)
    Language English
    Publishing date 2022-07-25
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021/acs.biochem.2c00270
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