Selective 2 '-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis

摘要

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPEAPEAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNARNARNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPEAPEAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPEAPEAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNARNARNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPEAPEAPE probing and to construct multiplexed SHAPEAPEAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPEAPEAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNARNARNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPEAPEAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNARNARNA motifs, rare components of complex RNARNARNA ensembles and entire transcriptomes.