Dissecting the energetic architecture within an RNA tertiary structural motif via high-throughput thermodynamic measurements

Abstract

Structured RNAs and RNA/protein complexes perform critical cellular functions. They often contain structurally conserved tertiary contact “motifs,” whose occurrence simplifies the RNA folding landscape. Prior studies have focused on the conformational and energetic modularity of intact motifs. Here, we turn to the dissection of one common motif, the 11nt receptor (11ntR), using quantitative analysis of RNA on a massively parallel array to measure the binding of all single and double 11ntR mutants to GAAA and GUAA tetraloops, thereby probing the energetic architecture of the motif. While the 11ntR behaves as a motif, its cooperativity is not absolute. Instead, we uncovered a gradient from high cooperativity amongst base-paired and neighboring residues to additivity between distant residues. As expected, substitutions at residues in direct contact with the GAAA tetraloop resulted in the largest decreases to binding, and energetic penalties of mutations were substantially smaller for binding to the alternate GUAA tetraloop, which lacks tertiary contacts present with the canonical GAAA tetraloop. However, we found that the energetic consequences of base partner substitutions are not, in general, simply described by base pair type or isostericity. We also found exceptions to the previously established stability–abundance relationship for 11ntR sequence variants. These findings of “exceptions to the rule” highlight the power of systematic high-throughput approaches to uncover novel variants for future study in addition to providing an energetic map of a functional RNA.

High-throughput determination of RNA tertiary contact thermodynamics by quantitative DMS chemical mapping

“…In a recent set of studies, Herschlag and colleagues characterized the binding affinities of approximately 3000 tetraloop receptor mutants to the GAAA tetraloop using the RNA-MaP platform (19, 21). They identified a subset of mutations with the same binding mode as the wild-type tetraloop-receptor (11ntR).…”

“…As the number of structured RNAs with potential tertiary contacts rapidly grows, there is a critical need for high-throughput methods. Greenleaf and colleagues have recently developed the RNA on a massively parallel array (RNA-MaP) platform (19)(20)(21). This device can simultaneously measure the stability of thousands of unique RNA tertiary contacts through fluorescence intensity.…”

“…This device can simultaneously measure the stability of thousands of unique RNA tertiary contacts through fluorescence intensity. The RNA-MaP platform is a significant technological advance, leading to several important insights about tertiary contact formation (19)(20)(21). However, these devices are custom-built using existing Illumina sequencers and, thus, are not accessible to other scientists.…”

Exploring the energetic and conformational properties of the sequence space connecting naturally occurring RNA tetraloop receptor motifs

“…The 11ntR, IC3R, and Vc2R (Fig. 1 A-C) were chosen to anchor our exploration of the TLR landscape due to their extensive prior study (Costa and Michel 1995;Ikawa et al 1999Ikawa et al , 2001Weinberg et al 2007;Geary et al 2008;Roth and Breaker 2009;Smith et al 2010;Bisaria and Herschlag 2015;Bonilla et al 2017Bonilla et al , 2021Shin et al 2023). We sampled the landscape spanning these receptors by investigating each stepwise sequence of mutations from the 11ntR to the two other TLRs, focusing on the shortest mutational pathways between the TLR sequences.…”

“…1). In-depth mutational analyses revealed that the residues within the 11ntR exhibit a mix of cooperative and additive energetic properties, with cooperativity dominating proximal groups and additivity dominating groups that are distal in three-dimensional space in the TLR structure (Shin et al 2023).…”