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

Abstract

Chemical mapping is a widespread technique for structural analysis of nucleic acids in which a molecule's reactivity to different probes is quantified at single-nucleotide resolution and used to constrain structural modeling. This experimental framework has been extensively revisited in the past decade with new strategies for high-throughput read-outs, chemical modification, and rapid data analysis. Recently, we have coupled the technique to high-throughput mutagenesis. Point mutations of a base-paired nucleotide can lead to exposure of not only that nucleotide but also its interaction partner. Carrying out the mutation and mapping for the entire system gives an experimental approximation of the molecules contact map. Here, we give our in-house protocol for this mutate-and-map strategy, based on 96-well capillary electrophoresis, and we provide practical tips on interpreting the data to infer nucleic acid structure.Comment: 22 pages, 5 figure