Direct identification of base-paired RNA nucleotides by correlated chemical probing.
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.
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Andrey
A
Krokhotin
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Anthony M
AM
Mustoe
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Kevin M
KM
Weeks
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Nikolay V
NV
Dokholyan
D000465:Algorithms; D020029:Base Pairing; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000235:genetics*; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012984:Software
D009711:Nucleotides; D012313:RNA
DMS reactivity; RNA secondary structure; chemical probing; pseudoknots; structure prediction