RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP)

The combination of selective 2'-hydroxyl acylation of RNA with high-throughput sequencing of the transcribed cDNA allows identification of chemically modified sites as mutations in the sequence that then yield highly accurate secondary-structure models of the RNA. Many biological processes are RNA-mediated, but higher-order structures for most RNAs are unknown, which makes it difficult to understand how RNA structure governs function. Here we describe selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) that makes possible de novo and large-scale identification of RNA functional motifs. Sites of 2′-hydroxyl acylation by SHAPE are encoded as noncomplementary nucleotides during cDNA synthesis, as measured by massively parallel sequencing. SHAPE-MaP–guided modeling identified greater than 90% of accepted base pairs in complex RNAs of known structure, and we used it to define a new model for the HIV-1 RNA genome. The HIV-1 model contains all known structured motifs and previously unknown elements, including experimentally validated pseudoknots. SHAPE-MaP yields accurate and high-resolution secondary-structure models, enables analysis of low-abundance RNAs, disentangles sequence polymorphisms in single experiments and will ultimately democratize RNA-structure analysis.