Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE

icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N-3 followed by selective chemical enrichment of NAI-N-3-modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in similar to 5 d, and the sequencing and bioinformatics data analysis take an additional 4-5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNARNARNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models.