SHARC-seq: High throughput in vivo determination of 3D RNA structures and dynamics

RNA molecules not only carry genetic information like DNA, but also folds into exquisite 3D structures like proteins. Despite strong interests in RNA biology and their medical applications, RNA structure determination in vivo remained a long-standing problem. Here we developed a new technology to directly determine RNA structures in vivo, termed SHARC-seq. Applying SHARC-seq, spatial distances among nucleotides can be measured and used to rebuild in vivo RNA 3D structure and dynamics. Overall design: Cells and tissues are crosslinked with 5mM, 12.5mM and 25mM SHARC, and crosslinked RNA is extracted using new developed TNA method. Extracted total RNA were fragmented with RNase III. After fragmentation, proximity ligation and reverse crosslinking, RNA are converted into cDNA library for high-throughput sequencing. In vitro transcribed 1HR2 are crosslinked with 5mM SHARC. Crosslinked 1HR2 RNA were fragmented with RNase III. After fragmentation, proximity ligation and reverse crosslinking, RNA are converted into cDNA library for high-throughput sequencing.