دورية أكاديمية
أعرض في EDS

An optimized chemical-genetic method for cell-specific metabolic labeling of RNA.

التفاصيل البيبلوغرافية
العنوان: An optimized chemical-genetic method for cell-specific metabolic labeling of RNA.
المؤلفون: Nainar S; Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA., Cuthbert BJ; Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA., Lim NM; Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA., England WE; Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA., Ke K; Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA., Sophal K; Department of Chemistry, University of California, Irvine, Irvine, CA, USA., Quechol R; Department of Chemistry, University of California, Irvine, Irvine, CA, USA., Mobley DL; Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA.; Department of Chemistry, University of California, Irvine, Irvine, CA, USA., Goulding CW; Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA.; Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA., Spitale RC; Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA. rspitale@uci.edu.; Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA. rspitale@uci.edu.; Department of Chemistry, University of California, Irvine, Irvine, CA, USA. rspitale@uci.edu.
المصدر: Nature methods [Nat Methods] 2020 Mar; Vol. 17 (3), pp. 311-318. Date of Electronic Publication: 2020 Feb 03.
نوع المنشور: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.
اللغة: English
بيانات الدورية: Publisher: Nature Pub. Group Country of Publication: United States NLM ID: 101215604 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1548-7105 (Electronic) Linking ISSN: 15487091 NLM ISO Abbreviation: Nat Methods Subsets: MEDLINE
أسماء مطبوعة: Original Publication: New York, NY : Nature Pub. Group, c2004-
مواضيع طبية MeSH: RNA/*chemistry , Uracil/*chemistry, Animals ; Azides/chemistry ; Biotinylation ; Catalytic Domain ; Coculture Techniques ; Deoxyuridine/analogs & derivatives ; Deoxyuridine/chemistry ; HEK293 Cells ; HeLa Cells ; Humans ; Kinetics ; Mice ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; NIH 3T3 Cells ; Nucleoside-Phosphate Kinase/metabolism ; Protein Domains ; RNA, Small Interfering/genetics ; Uridine/chemistry ; Uridine Kinase/metabolism
مستخلص: Tissues and organs are composed of diverse cell types, which poses a major challenge for cell-type-specific profiling of gene expression. Current metabolic labeling methods rely on exogenous pyrimidine analogs that are only incorporated into RNA in cells expressing an exogenous enzyme. This approach assumes that off-target cells cannot incorporate these analogs. We disprove this assumption and identify and characterize the enzymatic pathways responsible for high background incorporation. We demonstrate that mammalian cells can incorporate uracil analogs and characterize the enzymatic pathways responsible for high background incorporation. To overcome these limitations, we developed a new small molecule-enzyme pair consisting of uridine/cytidine kinase 2 and 2'-azidouridine. We demonstrate that 2'-azidouridine is only incorporated in cells expressing uridine/cytidine kinase 2 and characterize selectivity mechanisms using molecular dynamics and X-ray crystallography. Furthermore, this pair can be used to purify and track RNA from specific cellular populations, making it ideal for high-resolution cell-specific RNA labeling. Overall, these results reveal new aspects of mammalian salvage pathways and serve as a new benchmark for designing, characterizing and evaluating methodologies for cell-specific labeling of biomolecules.
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معلومات مُعتمدة: T34 GM069337 United States GM NIGMS NIH HHS; 1DP2GM119164 International U.S. Department of Health & Human Services | National Institutes of Health (NIH); R01 GM108889 United States GM NIGMS NIH HHS; DP2 GM119164 United States GM NIGMS NIH HHS; S10 RR025496 United States RR NCRR NIH HHS; R21 MH113062 United States MH NIMH NIH HHS; S10 OD010794 United States OD NIH HHS; P30 CA062203 United States CA NCI NIH HHS; 5R21MH113062 International U.S. Department of Health & Human Services | National Institutes of Health (NIH); R25 GM055246 United States GM NIGMS NIH HHS; S10 OD021718 United States OD NIH HHS; 1R01GM108889-01 International U.S. Department of Health & Human Services | National Institutes of Health (NIH)
المشرفين على المادة: 0 (Azides)
0 (RNA, Small Interfering)
26929-65-7 (2'-azido-2'-deoxyuridine)
56HH86ZVCT (Uracil)
63231-63-0 (RNA)
EC 2.7.1.48 (UCK2 protein, human)
EC 2.7.1.48 (Uridine Kinase)
EC 2.7.4.14 (cytidylate kinase)
EC 2.7.4.4 (Nucleoside-Phosphate Kinase)
W78I7AY22C (Deoxyuridine)
WHI7HQ7H85 (Uridine)
تواريخ الأحداث: Date Created: 20200205 Date Completed: 20200417 Latest Revision: 20211022
رمز التحديث: 20231215
مُعرف محوري في PubMed: PMC8518020
DOI: 10.1038/s41592-019-0726-y
PMID: 32015544
قاعدة البيانات: MEDLINE
الوصف
تدمد:1548-7105
DOI:10.1038/s41592-019-0726-y