The Mechanisms of RNA SHAPE Chemistry

Abstract

The biological functions of RNA are ultimately governed by the local environment at each nucleotide. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful approach for measuring nucleotide structure and dynamics in diverse biological environments. SHAPE reagents acylate the 2′-hydroxyl group at flexible nucleotides because unconstrained nucleotides preferentially sample rare conformations that enhance the nucleophilicity of the 2′-hydroxyl. The critical corollary is that some constrained nucleotides must be poised for efficient reaction at the 2′-hydroxyl group. To identify such nucleotides, we performed SHAPE on intact crystals of the E. coli ribosome, monitored the reactivity of 1490 nucleotides in 16S ribosomal RNA, and examined those nucleotides that were hyper-reactive towards SHAPE and had well-defined crystallographic conformations. Analysis of these conformations revealed that 2′-hydroxyl reactivity is broadly facilitated by general base catalysis involving multiple RNA functional groups and by two specific orientations of the bridging 3′-phosphate group. Nucleotide analog studies confirmed the contributions of these mechanisms to SHAPE reactivity. These results provide a strong mechanistic explanation for the relationship between SHAPE reactivity and local RNA dynamics and will facilitate interpretation of SHAPE information in the many technologies that make use of this chemistry.

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

“…Second, several nucleotides that are well-ordered in the small ribosomal crystal structure have been shown to be hyper-reactive to SHAPE reagents. This unexpectedly high reactivity was attributed to local effects such as potential general base catalysis by RNA functional groups and specific orientations of the flanking 3 ′ phosphate group (McGinnis et al 2012), which may occur in gene 60 mRNA as well. Third, Tb 3+ ions bind preferentially to and can displace metals from cation binding sites (Hargittai and Musier-Forsyth 2000;Walter et al 2000).…”

Structural basis for diversity in the SAM clan of riboswitches

“…To assess the influence of SAM binding on the structure of the env87 SAM-I/IV aptamer domain, selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing (26) was performed in the absence and presence of 1 mM SAM. SHAPE probing employs N-methylisotoic anhydride that generally reacts with 2′-hydroxyl groups of ribose sugars in conformationally dynamic regions of the backbone or where the hydroxyl group is locked into a conformation favoring in-line attack with the neighboring phosphodiester linkage (27). For each nucleotide, normalized reactivity in the presence of 1 mM SAM was subtracted from the normalized reactivity in the absence of SAM to yield a reactivity difference plot (Fig.…”

QuShape: Rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis

“…In the SHAPE (selective 29-hydroxyl acylation analyzed by primer extension) technologies, an RNA is reacted with an electrophilic reagent that can form an adduct with ribose 29-OH groups in a manner dependent on the conformational flexibility of each nucleotide ( Fig. 1A; Merino et al 2005;Gherghe et al 2008;McGinnis et al 2012). Sites in the RNA that form 29-O-adducts can be detected as stops to reverse transcriptase-mediated primer extension (Fig.…”

“…1C; Wilkinson et al 2008;McGinnis et al 2009;Watts et al 2009). SHAPE reactivities correlate strongly with model-free measurements of molecular order and are largely independent of nucleotide type or solvent accessibility (Gherghe et al 2008;Wilkinson et al 2009;McGinnis et al 2012). SHAPE reactivity information has been used to develop RNA secondary structure models, to detect changes in RNA conformation, and to monitor interactions with proteins, ligands, and metal ions.…”