Fingerprinting Noncanonical
and Tertiary RNA Structures
by Differential SHAPE Reactivity
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Abstract
Many RNA structures are composed of simple secondary
structure
elements linked by a few critical tertiary interactions. SHAPE chemistry
has made interrogation of RNA dynamics at single-nucleotide resolution
straightforward. However, de novo identification of nucleotides involved
in tertiary interactions remains a challenge. Here we show that nucleotides
that form noncanonical or tertiary contacts can be detected by comparing
information obtained using two SHAPE reagents, <i>N</i>-methylisatoic
anhydride (NMIA) and 1-methyl-6-nitroisatoic anhydride (1M6). Nucleotides
that react preferentially with NMIA exhibit slow local nucleotide
dynamics and usually adopt the less common C2′-endo ribose
conformation. Experiments and first-principles calculations show that
1M6 reacts preferentially with nucleotides in which one face of the
nucleobase allows an unhindered stacking interaction with the reagent.
Differential SHAPE reactivities were used to detect noncanonical and
tertiary interactions in four RNAs with diverse structures and to
identify preformed noncanonical interactions in partially folded RNAs.
Differential SHAPE reactivity analysis will enable experimentally
concise, large-scale identification of tertiary structure elements
and ligand binding sites in complex RNAs and in diverse biological
environments