Systematic Mutation and Unnatural Base Pair Incorporation Improves Riboswitch-Based Biosensor Response Time

Abstract

Engineered RNAs have applications in diverse fields from biomedical to environmental. In many cases, the folding of the RNA is critical to its function. Here we describe a strategy to improve the response time of a riboswitch-based fluorescent biosensor. Systematic mutagenesis was performed to either make transpose or transition base pair mutants or introduce orthogonal base pairs. Both natural and unnatural base pair mutants were found to improve the biosensor response time without compromising fold turn-on or ligand affinity. These strategies can be transferred to improve the performance of other RNA-based tools.

Keywords: RNA-based fluorescent biosensors; base pair mutagenesis; guanidine; orthogonal base pairs; rapid sensing; riboswitch folding.

Figure 1

(A) Schematic representation of Dru J 2,2-d-guanidine biosensor. (B) In vitro kinetic screening results of 34 single mutants. Results for each base pair are shown for the transpose mutation (top region) and transition mutation (bottom region). (C) In vitro turn-on kinetic plots of the WT and C4-G46 biosensors. Scattered dots represent the data points, and solid lines represent best-fit curves.

Figure 2

(A) Site-specific incorporation of Ds and Pa/Pa′ by in vitro transcription using synthetic DNA templates. (B) Fold turn-on of the WT and UBP containing biosensors. (C) In vitro turn-on kinetic plots of the WT biosensor and Pa3-Ds47 mutant. Scattered dots represent the data points, and solid lines represent best-fit curve.