Establishing an In Vivo RNA Accessibility Probing System and Transitioning to High-Throughput Characterization

Authors: Praveen Vimal¹, Jorge Vázquez-Anderson¹, and Lydia M. Contreras¹

¹McKetta Department of Chemical Engineering at the University of Texas at Austin, Austin, Texas 78712, USA

Abstract

RNA regulation controls features such as bacterial virulence and even tumor growth. RNA exerts its regulatory functions when interacting with other biomolecules (RNAs, proteins, and DNAs). This interaction heavily relies on its structure. Disruption of its structure can cause detrimental impacts on RNAs’ function. For this reason, it becomes critical to understand RNA structure. Scientists have developed a myriad of techniques to study RNA in vitro, requiring to extract the RNA from its native environment. While these techniques reveal the structure in a foreign environment, they do not show the relevant influence of intracellular factors. To this end a few efforts have been made to develop in vivo techniques that usually rely on the use of a foreign chemical.

We have developed a system that characterizes RNA structure in vivo (without a foreign chemical) providing a realistic measure of RNA accessibility for regulatory purposes. Our system is called the in vivo RNA Structural Sensing System (iRS³) and it measures the ability of a given target RNA region to establish base-pairing interactions with another RNA. The iRS³ has four different important parts: the probe, cis-blocking region (CB), ribosomal binding site (RBS), and green fluorescence protein (GFP) sequence downstream. When the targeted region is accessible the RBS is free from its interaction from the CB allowing translation of GFP. On the other hand when the targeted region is inaccessible the RBS remains sequestered by the CB preventing fluorescence generation. Hereby we show that fluorescence is a direct quantifiable approach to determine accessibility of any particular sequence on the target RNA molecule showing a relative capacity to be regulated by other RNAs. This data can be collected over several different probes to fully map the target RNA’s accessibility in vivo. We believe this information is instrumental in the design of antisense strategies to control expression. Finally we present a new approach to expand the current system to high-throughput application with the prospects of characterizing RNA accessibility in a genome-wide manner.