Optimized photochemistry and enzymology enable efficient analysis of RNA structures and interactions in cells

RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Direct determination of RNA structures and interactions in living cells remain a major challenge in biology. Crosslinking and proximity-ligation-based approaches, such as PARIS (Psoralen Analysis of RNA Interactions and Structures), offer many advantages, such as the ability to directly capture complex RNA duplexes in cells with single molecule accuracy, near base pair resolution in high throughput and without size limit. Based on a systematic optimization of the photochemistry and enzymology, here we present PARIS2, a re-invented method for capturing RNA duplexes with >3000-fold improved efficiency. PARIS2 captures ribosome small subunit binding sites on mRNAs, thus reporting translation process. PARIS2 also captures the snRNP binding sites on various types of RNA targets. We applied PARIS2 to enterovirus D-68, a re-emerging viral pathogen associated with severe neurological symptoms and discovered dynamic/alternative conformations in the 5’UTR. The new photochemical and enzymological improvements and the PARIS2 method should be widely applicable to basic RNA research and RNA-related diseases. Cells and tissues are crosslinked with AMT/Amotosalen and crosslinked RNA is extracted using new developed TNA method. mRNA and EVD68 viral RNA are enriched using Poly(A)Purist™ MAG Kit (Invitrogen) and antisense oligos respectively. After fragmentation, proximity ligation and reverse crosslinking, RNA are converted into cDNA library for high-throughput sequencing.