Many RNA structures are comprised 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 non-canonical or tertiary contacts are detected by comparing information obtained using two SHAPE reagents, N-methylisatoic anhydride (NMIA) and 1-methyl-6-nitroisatoic anhydride (1M6). Nucleotides that react preferentially with NMIA exhibit slow local nucleotide dynamics and preferentially adopt the less common C2′-endo ribose conformation. Experiments and first-principle calculations show 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 non-canonical and tertiary interactions in four RNAs with diverse structures and to identify pre-formed non-canonical 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.

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 ~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. Here we quantify covalent adducts in RNA directly by adduct-inhibited exoribonuclease degradation. RNA 2′-O-adducts block processivity of a 3′→5′ exoribonuclease, RNase R, to produce fragments that terminate three nucleotides 3′ of the modification site. We analyzed the structure of the native thiamine pyrophosphate (TPP) riboswitch aptamer domain and identified large changes in local nucleotide dynamics and global RNA structure upon ligand binding. In addition to numerous changes that can be attributed to ligand recognition, we identify a single nucleotide bulge register shift, distant from the binding site, that stabilizes the ligand-bound structure. Selective 2′-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) should prove broadly useful for facile structural analysis of small non-coding RNAs and for RNAs that have functionally critical structures at their 5′ and 3′ ends.

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 (Selective 2′-Hydroxyl Acylation analyzed by Protection from Exoribonuclease) 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 E. 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.