Direct mapping of higher-order RNA tertiary interactions by SHAPE-JuMP

Higher-order structure governs function for many RNAs. However, discerning this structure, especially for large molecules, is a challenging and unresolved problem. Here, we present SHAPE-JuMP (selective 2-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) to interrogate through-space RNA tertiary interactions in a direct and high-throughput manner. A bi-functional small molecule is used to chemically link two interacting nucleotides that occur in close three-dimensional space in an RNA structure. The RNA crosslink site is then encoded in a single, direct step into a complementary DNA (cDNA) strand via an engineered reverse transcriptase that jumps across the crosslinked nucleotides. The resulting cDNA contains a deletion relative to the native RNA sequence, detectable by sequencing, thereby indicating the site of the crosslinked nucleotides. SHAPE-JuMP measures RNA tertiary structure proximity in an experimentally concise way across large RNA molecules at nanometer resolution. SHAPE-JuMP is especially effective at measuring interactions involved in multi-helix junctions and loop-to-helix packing, enables modeling the global fold of RNAs up to several hundred nucleotides in length, and is poised to enable in-solution structural interrogation and modeling of diverse complex RNAs.