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Abstract RNA molecules adopt complex structures that perform essential biological functions across all forms of life, making them promising candidates for therapeutic applications. However, our ability to design new RNA structures remains limited by an incomplete understanding of their folding principles. While global metrics such as the minimum free energy are widely used, they are at odds with naturally occurring structures and incompatible with established design rules. Here, we introduce local stability compensation (LSC), a principle that RNA folding is governed by the local balance between destabilizing loops and their stabilizing adjacent stems, challenging the focus on global energetic optimization. Analysis of over 100,000 RNA structures revealed that LSC signatures are particularly pronounced in bulges and their adjacent stems, with distinct patterns across different RNA families that align with their biological functions. To validate LSC experimentally, we systematically analyzed thousands of RNA variants using DMS chemical mapping. Our results demonstrate that stem folding, as measured by reactivity, correlates with LSC (R²= 0.458 for hairpin loops) and that instabilities show no significant effect on folding for distal stems. These findings demonstrate that LSC can be a guiding principle for understanding RNA function and for the rational design of custom RNAs. Graphical Abstract 

Abstract Structural plasticity is integral to RNA function; however, there are currently few methods to quantitatively resolve RNAs that have multiple structural states. NMR spectroscopy is a powerful approach for resolving conformational ensembles but is size-limited. Chemical probing is well-suited for large RNAs but provides limited structural and kinetics information. Here, we integrate the two approaches to visualize a two-state conformational ensemble for the central stem–loop 3 (SL3) of 7SK RNA, a critical element for 7SK RNA function in transcription regulation. We find that the SL3 distal end exchanges between two equally populated yet structurally distinct states in both isolated SL3 constructs and full-length 7SK RNA. We rationally designed constructs that lock SL3 into a single state and demonstrate that both chemical probing and NMR data fit to a linear combination of the two states. Comparison of vertebrate 7SK RNA sequences shows either or both states are highly conserved. These results provide new insights into 7SK RNA structural dynamics and demonstrate the utility of integrating chemical probing with NMR spectroscopy to gain quantitative insights into RNA conformational ensembles.