In Vivo RNA Structure Probing with DMS-MaPseq.

Abstract

RNA has an extraordinary capacity to fold and form intrinsic secondary structures that play a central role in maintaining its functionality. It is crucial to have ways to study RNA structures and identify their functions in their biological environment. In the last few decades, a number of different chemical probing methods have been used to study RNA secondary structure. Here, we present a dimethyl sulfate-based (DMS) chemical probing method coupled with Next Generation sequencing (DMS-MaPseq) to study RNA secondary structure in vivo.DMS modifies unpaired adenine and cytosine bases which are then converted to mutations/mismatches using a thermostable group II intron reverse transcriptase (TGIRT) and further analyzed using sequencing. We validated the technique in model systems ranging from Drosophila to human cell lines, thus increasing the technique's broad range of applications. DMS-MaPseq provides high quality data and can be used for both gene-targeted as well as genome-wide analysis.