High-throughput analysis of RNA tertiary structure and interactions

Homan, Philip John

High-throughput analysis of RNA tertiary structure and interactions Public Deposited

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March 21, 2019

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Homan, Philip John
- Affiliation: College of Arts and Sciences, Department of Chemistry

Abstract

The many important cellular functions of RNA molecules depend on formation of complex tertiary structures. Knowledge of the specific interactions that stabilize these structures is key to understanding the function of the RNA. Current methods to study RNA tertiary structures are limited. Biophysical methods that measure RNA structure produce high-resolution tertiary structures but are limited by the size of the RNA. Current biochemical methods are difficult to interpret and can only give an average view of all possible structures in solution. In this work I develop two new biochemical techniques that can be used to map tertiary interactions within RNA. First I develop a method in which I blend the principals from modification interference experiments with SHAPE chemistry called 2'-hydroxyl molecular interference (HMX). With this approach I am able to measure structurally crowded regions of an RNA and incorporate this data as experimental constraints in discrete molecular dynamics simulations to yield experimentally informed, three-dimensional models. This method was developed with a small test set of RNA and applied to the Tetrahymena group I intron to identify interactions between multiple substructures in the RNA. Second I develop a method termed RING-MaP in which I utilize multi-nucleotide sequencing to detect multiple chemical modifications in a single RNA molecule in order to identify correlated structural interactions between modified nucleotides. With this technique I am able to map through-space interaction networks in RNA of increasing size. Furthermore, through the use of spectral clustering analysis, I am able to identify multiple structural conformations in a single, in-solution ensemble of RNA.

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