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

Autor: 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.
Jazyk: angličtina
Zdroj: Nature methods [Nat Methods] 2020 Mar; Vol. 17 (3), pp. 311-318. Date of Electronic Publication: 2020 Feb 03.
DOI: 10.1038/s41592-019-0726-y
Abstrakt: 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.
Databáze: MEDLINE
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