Changing the Ligand Specificity of a Riboswitch from Guanine to Hypoxanthine

Abstract

Riboswitches are RNA elements in messenger RNAs that regulate gene expression by undergoing a ligand‐triggered conformational change. Previously, we modified several riboswitches for use as sensitive and specific sensors for their ligands. To extend the utility of our sensor design, we developed an in vitro selection strategy to isolate variants of riboswitches that have altered ligand specificity. We initially tested our strategy by attempting to change the specificity of a guanine riboswitch from guanine to hypoxanthine. Our first selection experiment produced only the original, wild‐type guanine riboswitch. In hindsight, this was not surprising since the guanine riboswitch can detect hypoxanthine (albeit poorly), and it was the most abundant RNA in our initial partially‐randomized RNA pool. For our second experiment, we reduced the abundance of the wild‐type riboswitch by increasing the degree of sequence randomization. This experiment resulted in isolating RNAs with many sequence changes and deletions that failed to detect hypoxanthine. We reasoned that these aberrant RNAs arose during PCR with Taq DNA polymerase. Here, we describe the results of a new selection experiment in which we used a proofreading DNA polymerase and a redesigned partially‐randomized RNA pool.Support or Funding InformationDefense Threat Reduction Agency (DTRA)Office of Naval Research (ONR)Chemistry Department, United States Naval AcademyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.