Chemical crosslinking and ligation methods for in vivo analysis of RNA structures and interactions.

RNA structures and interactions in living cells drive a variety of biological processes and play critical roles in physiology and disease states. However, studies of RNA structures and interactions have been challenging due to limitations in available technologies. Direct determination of structures in vitro has been only possible to a small number of RNAs with limited sizes and conformations. We recently introduced two chemical crosslink-ligation techniques that enabled studies of transcriptome-wide secondary and tertiary structures and their dynamics. In a dramatically improved version of the psoralen analysis of RNA interactions and structures (PARIS2) method, we detailed the synthesis and use of amotosalen, a highly soluble psoralen analogue, and enhanced enzymology for higher efficiency duplex capture. We also introduced spatial 2'-hydroxyl acylation reversible crosslinking (SHARC) with exonuclease (exo) trimming, a method which utilizes a novel crosslinker class that targets the 2'-OH to capture three-dimensional (3D) structures. Both are powerful orthogonal approaches for solving in vivo RNA structure and interactions, integrating crosslinking, exo trimming, proximity ligation, and high throughput sequencing. In this chapter, we present a detailed protocol for the methods and highlight steps that outperform existing crosslink-ligation approaches.

Chemical reversible crosslinking enables measurement of RNA 3D distances and alternative conformations in cells.

Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a method, Spatial 2'-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA. Integrating crosslinking, exonuclease (exo) trimming, proximity ligation, and high throughput sequencing, SHARC enables transcriptome-wide tertiary structure contact maps at high accuracy and precision, revealing heterogeneous RNA structures and interactions. SHARC data provide constraints that improves Rosetta-based RNA 3D structure modeling at near-nanometer resolution. Integrating SHARC-exo with other crosslinking-based methods, we discover compact folding of the 7SK RNA, a critical regulator of transcriptional elongation. These results establish a strategy for measuring RNA 3D distances and alternative conformations in their native cellular context.

Optimized photochemistry enables efficient analysis of dynamic RNA structuromes and interactomes in genetic and infectious diseases.

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (-) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases.