Fingerprinting noncanonical and tertiary RNA structures by differential SHAPE reactivity.

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.

Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA.

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.

Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a powerful approach for characterizing RNA structure and dynamics at single-nucleotide resolution. However, SHAPE technology is limited, sometimes severely, because primer extension detection obscures structural information for approximately 15 nts at the 5' end and 40-60 nts at the 3' end of the RNA. Moreover, detection by primer extension is more complex than the actual structure-selective chemical interrogation step.