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Abstract Structured RNAs often contain long-range tertiary contacts that are critical to their function. Despite the importance of tertiary contacts, methods to measure their thermodynamics are low throughput or require specialized instruments. Here, we introduce a new quantitative chemical mapping method (qMaPseq) to measure Mg2+-induced formation of tertiary contact thermodynamics in a high-throughput manner using standard biochemistry equipment. With qMaPseq, we measured the ΔG of 98 unique tetraloop/tetraloop receptor (TL/TLR) variants in a one-pot reaction. These results agree well with measurements from specialized instruments (R2= 0.64). Furthermore, the DMS reactivity of the TL directly correlates to the stability of the contact (R2= 0.68), the first direct evidence that a single DMS reactivity measurement reports on thermodynamics. Combined with structure prediction, DMS reactivity allowed the development of experimentally accurate 3D models of TLR mutants. These results demonstrate that qMaPseq is broadly accessible, high-throughput and directly links DMS reactivity to thermodynamics. 

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.