Mutation signature filtering enables high-fidelity RNA structure probing at all four nucleobases with DMS.

Chemical probing experiments have transformed RNA structure analysis, enabling high-throughput measurement of base-pairing in living cells. Dimethyl sulfate (DMS) is one of the most widely used structure probing reagents and has played a pivotal role in enabling next-generation single-molecule probing analyses. However, DMS has traditionally only been able to probe adenine and cytosine nucleobases. We previously showed that, using appropriate conditions, DMS can also be used to interrogate base-pairing of uracil and guanines in vitro at reduced accuracy. However, DMS remained unable to informatively probe guanines in cells. Here, we develop an improved DMS mutational profiling (MaP) strategy that leverages the unique mutational signature of N1-methylguanine DMS modifications to enable high-fidelity structure probing at all four nucleotides, including in cells. Using information theory, we show that four-base DMS reactivities convey greater structural information than current two-base DMS and SHAPE probing strategies. Four-base DMS experiments further enable improved direct base-pair detection by single-molecule PAIR analysis, and ultimately support RNA structure modeling at superior accuracy. Four-base DMS probing experiments are straightforward to perform and will broadly facilitate improved RNA structural analysis in living cells.