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

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 (qDMS-MaPseq) to measure Mg ²⁺ -induced formation of tertiary contact thermodynamics in a high-throughput manner using standard biochemistry equipment. With qDMS-MaPseq, 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 (R ² =0.64). Furthermore, the DMS reactivity of the TL directly correlates to the stability of the contact (R ² =0.68), the first direct evidence that DMS reactivity reports on thermodynamics. Combined with structure prediction, DMS reactivity allowed the development of experimentally accurate 3D models of TLR mutants. These results demonstrate that qDMS-MaPseq is broadly accessible, high-throughput, and directly links DMS reactivity to thermodynamics.