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Chemical mapping is a widespread technique for structural analysis of nucleicacids in which a molecule's reactivity to different probes is quantified atsingle-nucleotide resolution and used to constrain structural modeling. Thisexperimental framework has been extensively revisited in the past decade withnew strategies for high-throughput read-outs, chemical modification, and rapiddata analysis. Recently, we have coupled the technique to high-throughputmutagenesis. Point mutations of a base-paired nucleotide can lead to exposureof not only that nucleotide but also its interaction partner. Carrying out themutation and mapping for the entire system gives an experimental approximationof the molecules contact map. Here, we give our in-house protocol for thismutate-and-map strategy, based on 96-well capillary electrophoresis, and weprovide practical tips on interpreting the data to infer nucleic acidstructure.

A mutate-and-map protocol for inferring base pairs in structured RNA