(A) s4U disulfide exchange with HPDP-biotin. (B) LC-MS extracted ion
chromatograms of s4U (red) and biotin-s4U (blue) for
HPDP-biotin at the indicated reaction times. (C) s4U disulfide
exchange with MTS-biotin. (D) LC-MS chromatograms as in (B). (E) Downfield
1H NMR spectra of (top) s4U alone, (center)
s4U reacted with 3-[2-Pyridyldithio]propionyl
hydrazide (PDPH), an HPDP-like disulfide, and (bottom) methyl-MTS. Peaks for the
starting material (red shading) and products (blue shading) were integrated and
normalized to sum of the anomeric protons of s4U and its products
(5.9 ppm). For full spectra, see Figure S1A–C. (F, G)
Enrichment of a singly-thiolated 39-nt RNA by (F) HPDP-biotin or (G) MTS-biotin.
Fluorescently labeled 39-nt RNAs with or without a single s4U were
biotinylated with the indicated reagent and enriched on streptavidin beads,
followed by urea-PAGE and fluorescence imaging. See also Figure S1.