Changing the Ligand Specificity of a Guanine Riboswitch by In Vitro Selection
Abstract
Riboswitches are RNA elements in messenger RNAs that regulate gene expression by undergoing a ligand‐triggered conformational change. Riboswitches bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. We previously modified several purine riboswitches for use in vitro and, indeed, they could detect their ligands with high sensitivity and specificity (1). 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 are attempting to find variants of the guanine riboswitch that can detect hypoxanthine rather than guanine. This is a challenging task because hypoxanthine is identical to guanine except for the absence of an exocyclic amino group. We partially randomized the sequence of our guanine sensor, and used hypoxanthine as the ligand during selection. Our first 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. To solve this problem we designed a new experiment based on the C74U mutant of the guanine riboswitch. C74 forms a Watson‐Crick base pair with the bound guanine ligand. Changing C74 to U results in a riboswitch that cannot bind to guanine or hypoxanthine but can bind weakly to adenine (2). We performed a second selection in which we partially randomized the sequence of the C74U mutant, except every variant retained a U at position 74. Since the C74U mutant cannot bind to hypoxanthine, we reasoned that we could select variants that could do so by allowing hypoxanthine to form a “wobble” pair with U74. We also anticipated that we could isolate hypoxanthine sensors that discriminate against guanine due to steric clashes between the sensor and the exocyclic amino group of guanine. After 11 rounds of selection, the selected RNA pool could no longer detect adenine but could detect hypoxanthine and guanine. The sensitivity for hypoxanthine and guanine was much lower than that of the wild‐type guanine riboswitch. However, the selectivity for hypoxanthine versus guanine was slightly improved, suggesting that the pool may contain individual variants that can discriminate against guanine. We are currently determining the sequence of a few variants in the selected pool to identify RNAs that we would like to test individually for their ability to specifically detect hypoxanthine.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Apr 01, 2019
- Source ID
- 10.1096/fasebj.2019.33.1_supplement.625.1
Entities
People
- Daniel Paul Morse
- Hasan P Abdullah
Organizations
- Defense Threat Reduction Agency
- Office of Naval Research
- United States Naval Academy