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Mol Cell. 2015 Sep 3;59(5):858-66. doi: 10.1016/j.molcel.2015.07.023.

Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry.

Author information

1
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA.
2
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA.
3
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA. Electronic address: matthew.simon@yale.edu.

Abstract

We describe a chemical method to label and purify 4-thiouridine (s(4)U)-containing RNA. We demonstrate that methanethiosulfonate (MTS) reagents form disulfide bonds with s(4)U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s(4)U labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA, such as 4-thiouridine tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover.

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PMID:
26340425
PMCID:
PMC4560836
DOI:
10.1016/j.molcel.2015.07.023
[Indexed for MEDLINE]
Free PMC Article

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