High-throughput dissection of the thermodynamic and conformational properties of a ubiquitous class of RNA tertiary contact motifs

Abstract

Despite RNA’s diverse secondary and tertiary structures and its complex conformational changes, nature utilizes a limited set of structural “motifs”—helices, junctions, and tertiary contact modules—to build diverse functional RNAs. Thus, in-depth descriptions of a relatively small universe of RNA motifs may lead to predictive models of RNA tertiary conformational landscapes. Motifs may have different properties depending on sequence and secondary structure, giving rise to subclasses that expand the universe of RNA building blocks. Yet we know very little about motif subclasses, given the challenges in mapping conformational properties in high throughput. Previously, we used “RNA on a massively parallel array” (RNA-MaP), a quantitative, high-throughput technique, to study thousands of helices and two-way junctions. Here, we adapt RNA-MaP to study the thermodynamic and conformational properties of tetraloop/tetraloop receptor (TL/TLR) tertiary contact motifs, analyzing 1,493 TLR sequences from different classes. Clustering analyses revealed variability in TL specificity, stability, and conformational behavior. Nevertheless, natural GAAA/11ntR TL/TLRs, while varying in tertiary stability by ∼2.5 kcal/mol, exhibited conserved TL specificity and conformational properties. Thus, RNAs may tune stability without altering the overall structure of these TL/TLRs. Furthermore, their stability correlated with natural frequency, suggesting thermodynamics as the dominant selection pressure. In contrast, other TL/TLRs displayed heterogenous conformational behavior and appear to not be under strong thermodynamic selection. Our results build toward a generalizable model of RNA-folding thermodynamics based on the properties of isolated motifs, and our characterized TL/TLR library can be used to engineer RNAs with predictable thermodynamic and conformational behavior.

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

“…Our results show much weaker binding of the 11ntR to the GUAA tetraloop (relative to GAAA), in agreement with prior data (ΔΔG = 3.5 kcal/mol, black dashed line in Fig. 3B; (13,40)). This large effect presumably arises because A2ʹ of the tetraloop, which makes multiple direct interactions with the 11ntR, is mutated to a U, thereby removing several direct tertiary interactions (Fig.…”

“…Indeed, Bonilla et al . (40) found that the correlation increased for the 70 11ntR variants observed in nature under more biologically relevant conditions, with lower Mg 2+ and K + present ( ρ = −0.47 vs . −0.69).…”

“…While the extensive energetic effects and energetic interrelationships strongly imply coupled conformational effects within the 11ntR • GAAA, the orientations of the helices that emanate from this motif appear to be unchanged based on the observation of indistinguishable thermodynamic fingerprints for measurable mutants ( SI Appendix Fig. S5 A ; (36, 40)). The five scaffolds studied provide a range of approach orientations for the 11ntR • GAAA interaction, and the mutants follow the same affinity pattern as wild-type across the scaffolds, despite weaker overall binding.…”

“…We analyzed a subset of natural 11ntR variants presented in Bonilla et al (40), which established an exponential correlation between abundance and stability. This correlation makes predictions for the abundance of variants based on their stabilities, including variants that are not observed in nature.…”

“…Pertinent to this study, the high-throughput nature of RNA-MaP enabled exploration of thousands of tetraloop receptors (TLRs) in different local structural contexts, as described in Bonilla et al . (40). In brief, Bonilla et al .…”