In Vitro Selection of RNAs That Can Detect Hypoxanthine: A Surprising Technical Issue

Abstract

The goal of this project is to find RNA molecules that detect hypoxanthine with great sensitivity and specificity. Hypoxanthine is the product of a family of RNA editing enzymes called ADARs. A sensor that can detect hypoxanthine will facilitate the study of the regulation of ADAR activity. There are naturally occurring guanine sensors called guanine riboswitches. We are using in vitroselection to find variants of a guanine riboswitch that can detect hypoxanthine rather than guanine. While performing the selection experiment, we encountered a technical problem. Instead of isolating hypoxanthine sensors, a group of related aberrant RNAs dominated the selected RNA pool. These RNAs had many sequence changes and deletions compared to the sequence of the guanine riboswitch, and they could not detect hypoxanthine. The aberrant RNAs were produced not by the selection process, but as a consequence of multiple rounds of amplification. Here we report our attempts to understand why this happened and to prevent the formation of the aberrant RNAs.