Chemical probing of RNA tertiary structure in a whole transcriptome at single-atom resolution

Trustees Of Boston University, Boston

Investigators

Abstract

RNA (ribonucleic acid) is now widely appreciated to be a key participant in nearly all of the activities of the cell, yet only limited information on the three-dimensional structures of most RNA molecules are available. This presents a challenge, because it is known that the biological function of an RNA molecule depends on its structure. To understand the relationship between structure and function for the large number of RNA molecules that populate the cell, new high-throughput experimental approaches for structure determination are needed. This project will develop a new experimental method that can provide detailed information on the three-dimensional folding of all the RNA molecules in a cell, in one experiment. The new method combines chemical probing of RNA structure by the hydroxyl radical, the quintessential Reactive Oxygen Species, with analysis by high-throughput sequencing, so that the structures of all RNA molecules in a cell can be monitored simultaneously. This proposal will provide educational and training opportunities for graduate and undergraduate students including underrepresented groups in STEM fields. The aim of this project is to develop a new method for obtaining tertiary structural information for all of the RNA in a cell, in one experiment. The experimental approach will adapt the hydroxyl radical chemical probing experiment for analysis by high-throughput sequencing. A key innovation will be the synthesis of a novel set of "catch and release" small molecule probes that will be used to capture the major product of hydroxyl radical cleavage of RNA, a strand terminated by a 5'-aldehyde moiety. Hydroxyl radical cleavage and aldehyde catch and release will be performed in flow format, to streamline the method. The method will be developed and validated by applying it to RNA molecules of known structure. The validated method will then be used to map the tertiary structure of the entire transcriptome, in vitro and in living cells, for yeast and human cells. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.