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      Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution.

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          Abstract

          Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2'-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized primer extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100-200 nucleotides.

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          Author and article information

          Journal
          Nat Protoc
          Nature protocols
          Springer Science and Business Media LLC
          1750-2799
          1750-2799
          2006
          : 1
          : 3
          Affiliations
          [1 ] Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
          Article
          nprot.2006.249
          10.1038/nprot.2006.249
          17406453
          6340377f-14cf-4c2b-a080-8c41952bfa16
          History

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