Why probe RNA with chemicals? RNAs fold into elaborate structures that are important for their functions. These structures can influence RNA stability, protein binding, sense the concentrations of small molecules and in extreme cases can even allow catalysis. One classic method to gain insight into RNA structure and function is to treat RNA with reactive molecules that react with some regions of the RNA and not others. Most commonly these reagents (such as SHAPE reagents, DMS or CMC) react with RNA that is flexible or single stranded. Identifying which nucleotides react and which do not can provide important constraints that are useful to predict RNA structure or to reveal regions of RNA that have different accessibility in different biological contexts.
Targeted Structure-Seq. Although Xist was first reported in 1990 and has since been studied by many groups, very little is known about the biochemical details that are responsible for the biological activity of this RNA. Open questions include how the RNA binds to chromatin, how Xist molecules can specifically spread in cis across mega-bases of DNA, and what biochemical activities allow it to silence genes. Recognizing that Xist structure is likely important for its function, we adapted chemical probing approaches to map the conformation of Xist in cells (Fang, Moss, et al., PLoS Genetics, 2015). This work was the first chemical probing of a cellular RNA of this size, and revealed intricate motifs spread across the RNA that may be responsible for some of the biochemical activities of Xist.
Reverse transcriptase induced mutations and termination events. Targeted Structure-seq was developed using inspiration from other approaches (e.g., Structure Seq and DMS-seq) to chemically probe RNAs using a sequencing output. In these cases, the location of the modified bases is generally read out by reverse transcriptase termination events induced by the chemical adduct. Instead of using termination events to map modification sites, an alternative approach is to use mutations in a sequencing experiment to map where the RNA is modified. We undertook a systematic comparison of these two approaches. We discovered that these two readouts are not well-correlated (Sexton et al., Biochemistry, 2017), which was quite surprising given that the field was using these readouts as if they were interchangeable. Instead, mutation events and termination events offer complementary information, thereby providing a more complete picture of accessible nucleotides in an RNA and enhancing RNA chemical probing experiments.
Development of a EDC to probe G and U nucleotides in cells. The most successful in cell probing reagents include SHAPE, which measures nucleotide flexibility, and DMS, which probes the accessibility of the Watson-Crick face of A and C nucleotides. While CMCT has long been used to probe G and U residues outside of cells, its bulky charged structure makes CMCT poorly suited for in cell probing. To address this shortcoming, we developed EDC as an alternative reagent to probe G and U nucleotides in cells (Wang et al. RNA, 2018).