Direct identification of base-paired RNA nucleotides by correlated chemical probing
- 1Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 2Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- Corresponding authors: weeks{at}unc.edu, dokh{at}unc.edu
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↵3 These authors contributed equally to this work.
Abstract
Many RNA molecules fold into complex secondary and tertiary structures that play critical roles in biological function. Among the best-established methods for examining RNA structure are chemical probing experiments, which can report on local nucleotide structure in a concise and extensible manner. While probing data are highly useful for inferring overall RNA secondary structure, these data do not directly measure through-space base-pairing interactions. We recently introduced an approach for single-molecule correlated chemical probing with dimethyl sulfate (DMS) that measures RNA interaction groups by mutational profiling (RING-MaP). RING-MaP experiments reveal diverse through-space interactions corresponding to both secondary and tertiary structure. Here we develop a framework for using RING-MaP data to directly and robustly identify canonical base pairs in RNA. When applied to three representative RNAs, this framework identified 20%–50% of accepted base pairs with a <10% false discovery rate, allowing detection of 88% of duplexes containing four or more base pairs, including pseudoknotted pairs. We further show that base pairs determined from RING-MaP analysis significantly improve secondary structure modeling. RING-MaP-based correlated chemical probing represents a direct, experimentally concise, and accurate approach for detection of individual base pairs and helices and should greatly facilitate structure modeling for complex RNAs.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.058586.116.
- Received August 3, 2016.
- Accepted October 28, 2016.
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