title: Enriching s4U-RNA Using Methane Thiosulfonate (MTS) Chemistry.
abstract: Metabolic labeling of cellular RNA is a useful approach to study RNA biology. 4-Thiouridine (s4U) is a convenient nucleoside for metabolic labeling because it is cell permeable and is incorporated into newly transcribed RNA, and the sulfur moiety provides a handle for biochemical purification. However, a critical step in the purification of s4U-RNA is the efficiency of the chemistry used to enrich s4U-RNA. Here, we present a protocol for s4U-RNA enrichment that includes efficient and reversible covalent chemistry to biotinylate s4U-RNA using the activated disulfide methane thiosulfonate conjugated to biotin (MTS-biotin), followed by enrichment on streptavidin beads. The efficiency of this chemistry reduces enrichment bias and requires less starting material, thereby expanding the utility of s4U to study RNA biology. © 2016 by John Wiley & Sons, Inc.
authors.affiliation: Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Duffy
authors.affiliation: Chemical Biology Institute, Yale University, West Haven, Connecticut.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Simon
mesh_terms: D001710:Biotin / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008698:Mesylates / Q000737:chemistry*; D012313:RNA / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry; D013891:Thiouridine / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D008698:Mesylates; D013891:Thiouridine; C074502:methanethiosulfonate; D012313:RNA; D001710:Biotin; D019809:Streptavidin
keywords: 4-thiouridine; RNA metabolism; metabolic labeling; methane thiosulfonate