RNA structure analysis at single nucleotide resolution by selective 2'-hydroxyl acylation and primer extension (SHAPE).
The reactivity of an RNA ribose hydroxyl is shown to be exquisitely sensitive to local nucleotide flexibility because a conformationally constrained adjacent 3'-phosphodiester inhibits formation of the deprotonated, nucleophilic oxyanion form of the 2'-hydroxyl group. Reaction with an appropriate electrophile, N-methylisatoic anhydride, to form a 2'-O-adduct thus can be used to monitor local structure at every nucleotide in an RNA. We develop a quantitative approach involving Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) to map the structure of and to distinguish fine differences in structure for tRNAAsp transcripts at single nucleotide resolution. Modest extensions of the SHAPE approach will allow RNA structure to be monitored comprehensively and at single nucleotide resolution for RNAs of arbitrary sequence and structural complexity and under diverse solution environments.
Merino
Edward J
EJ
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
Wilkinson
Kevin A
KA
Coughlan
Jennifer L
JL
Weeks
Kevin M
KM
D000215:Acylation; D000812:Anhydrides / Q000737:chemistry; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012349:RNA, Transfer, Asp / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012266:Ribose / Q000737:chemistry; D013329:Structure-Activity Relationship; D062367:ortho-Aminobenzoates / Q000737:chemistry
D000812:Anhydrides; C034477:RNA primers; D012349:RNA, Transfer, Asp; D062367:ortho-Aminobenzoates; C077537:N-methylisatoic anhydride; D012313:RNA; D012266:Ribose