In vivo SHAPE-seq: A method to determine the in-cell structure of RNA

Motivation · RNA is versatile, easy to design, and characterize [1] · RNAe regulate transcription, RNA degradation, and translation · SHAPE-Seq (elective 2'-ydroxyl ucylation analyzed by primer ntension puencing) is a powerful technique for characterizing RNA by determining structure [2], but can only measure in vitro transcribed RNA · We wish to know cellular RNA strscture to better understand their structure and function inside the cellular environment In-Cell SHAPE-Seq 1) Fluorescence assay allows discovering RNA regulator structure- function relationships 2) Modular platform allows probing almost any RNA with uniform set of primers Probing Results · Structure of cis-repreeeed (Cr) and trans-activating (ta) riboregulators differ slightly from proposed structure [3] · Ribosome binding site )RBS) nucleotide (nt) reactivity correlates well with fluorescence increase Conclusions · In-cell SHAPE-Seq is a powerful method to determine RNA structure with nucleotide resolution · Structural variations and increase in RBS nt reactivity were detected in cr and ta riboregulators Future Studies · Probe CRISPR (plustered regularly ntersperaed gpacer palindromic Lepeats) small guide RNA5 )5gRNA) to find structure- function relationships · Discover cellular structure, mechanism of small transcription activating RNA5 (STARs) [4] · Formulate design princtples for directed RNA engineering.