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.
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.