title: Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA.
abstract: RNA SHAPE chemistry yields quantitative, single-nucleotide resolution structural information based on the reaction of the 2'-hydroxyl group of conformationally flexible nucleotides with electrophilic SHAPE reagents. However, SHAPE technology has been limited by the requirement that sites of RNA modification be detected by primer extension. Primer extension results in loss of information at both the 5' and 3' ends of an RNA and requires multiple experimental steps. Here we describe RNase-detected SHAPE that uses a processive, 3'→5' exoribonuclease, RNase R, to detect covalent adducts in 5'-end-labeled RNA in a one-tube experiment. RNase R degrades RNA but stops quantitatively three and four nucleotides 3' of a nucleotide containing a covalent adduct at the ribose 2'-hydroxyl or the pairing face of a nucleobase, respectively. We illustrate this technology by characterizing ligand-induced folding for the aptamer domain of the Escherichia coli thiamine pyrophosphate riboswitch RNA. RNase-detected SHAPE is a facile, two-day approach that can be used to analyze diverse covalent adducts in any RNA molecule, including short RNAs not amenable to analysis by primer extension and RNAs with functionally important structures at their 5' or 3' ends.
authors: Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, USA.
authors: Kady-Ann
authors: KA
authors: Steen
authors: Nathan A
authors: NA
authors: Siegfried
authors: Kevin M
authors: KM
authors: Weeks
mesh_terms: D000215:Acylation; D052157:Aptamers, Nucleotide; D001483:Base Sequence; D018736:DNA Adducts; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005095:Exoribonucleases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008024:Ligands; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D059370:RNA Folding
chemical_list: D052157:Aptamers, Nucleotide; D018736:DNA Adducts; D008024:Ligands; D012313:RNA; D005095:Exoribonucleases; C080868:ribonuclease R