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

Chemical mapping is a widespread technique for structural analysis of nucleic acids in which a molecules 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.

Quantitative DMS mapping for automated RNA secondary structure inference

For decades, dimethyl sulfate (DMS) mapping has informed manual modeling of RNA structure in vitro and in vivo. Here, we incorporate DMS data into automated secondary structure inference using a pseudo-energy framework developed for 2-OH acylation (SHAPE) mapping. On six non-coding RNAs with crystallographic models, DMS- guided modeling achieves overall false negative and false discovery rates of 9.5% and 11.6%, comparable or better than SHAPE-guided modeling; and non-parametric bootstrapping provides straightforward confidence estimates. Integrating DMS/SHAPE data and including CMCT reactivities give small additional improvements. These results establish DMS mapping - an already routine technique - as a quantitative tool for unbiased RNA structure modeling.

The Stanford RNA Mapping Database for sharing and visualizing RNA structure mapping experiments

We have established an RNA Mapping Database (RMDB) to enable a new generation of structural, thermodynamic, and kinetic studies from quantitative single-nucleotide-resolution RNA structure mapping (freely available at http://rmdb.stanford.edu). Chemical and enzymatic mapping is a rapid, robust, and widespread approach to RNA characterization. Since its recent coupling with high-throughput sequencing techniques, accelerated software pipelines, and large-scale mutagenesis, the volume of mapping data has greatly increased, and there is a critical need for a database to enable sharing, visualization, and meta-analyses of these data. Through its on-line front-end, the RMDB allows users to explore single-nucleotide-resolution chemical accessibility data in heat-map, bar-graph, and colored secondary structure graphics; to leverage these data to generate secondary structure hypotheses; and to download the data in standardized and computer-friendly files, including the RDAT and community-consensus SNRNASM formats. At the time of writing, the database houses 38 entries, describing 2659 RNA sequences and comprising 355,084 data points, and is growing rapidly.