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Applying riboswitches for novel sensing and chemistry

  • Author(s): Truong, Johnny
  • Advisor(s): Hammond, Ming C
  • Francis, Matthew B
  • et al.
Abstract

Riboswitches are cis-regulatory structured RNA elements capable of controlling expression of downstream genes by binding to small molecule ligands. These naturally evolved RNA elements possess remarkable affinity and selectivity for their small molecule ligands, high folding efficiencies, and thermostability for functioning in cellular environments. Due to these properties, a number of riboswitch-based technologies have emerged such as riboswitch reporters, aptazymes, and RNA-based fluorescent (RBF) biosensors which all have wide applications for detection, imaging, and regulatory circuits. While riboswitch reporters and aptazymes have been robustly studied to better understand how to improve their function, there are fewer studies that expand on RBF biosensor development. Here, novel approaches towards expanding the functional repertoire of RBF biosensors and systematically probing their properties are described.

First, we show that engineering circular permutations of the riboswitch aptamer domain yields functional biosensors for S-adenosyl-L-methionine (SAM), using the SAM-I riboswitch as our model. We reveal that this design can enhance fluorescence turn-on and ligand binding affinity compared to the non-permuted topology. Expanding upon these established design principles, novel biosensors for the ligand guanidine was developed. Two novel designs were added to our existing repertoire that generated functional RBF biosensors using the architecture of the guanidine-I riboswitch. A new base-pair mutation strategy was applied on these guanidine biosensors which, resulting in modest changes to biosensor activation speeds just from single base-pair mutations. Lastly, riboswitches were explored as potent scaffolds to generate a self-labeling ribozyme. Various natural or engineered riboswitches for the electrophilic ligand, SAM, were screened for reactivity with an analog, Hey-SAM, as a proxy to measure ribozyme activity. In collaboration with Agilent Labs, a high-throughput method was developed for probing and screening latent ribozyme activity using a microarray platform. The efforts and strategies put forth here use riboswitches outside their native context for applications in detection and catalysis further showcasing the broad utility of riboswitch-based tools.

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