Guanidine Biosensors Enable Comparison of Cellular Turn-on Kinetics of Riboswitch-Based Biosensor and Reporter

Abstract

Cell-based sensors are useful for many synthetic biology applications, including regulatory circuits, metabolic engineering, and diagnostics. While considerable research efforts have been made toward recognizing new target ligands and increasing sensitivity, the analysis and optimization of turn-on kinetics is often neglected. For example, to our knowledge there has been no systematic study that compared the performance of a riboswitch-based biosensor versus reporter for the same ligand. In this study, we show the development of RNA-based fluorescent (RBF) biosensors for guanidine, a common chaotropic agent that is a precursor to both fertilizer and explosive compounds. Guanidine is cell permeable and nontoxic to E. coli at millimolar concentrations, which in contrast to prior studies enabled direct activation of the riboswitch-based biosensor and corresponding reporter with ligand addition to cells. Our results reveal that the biosensors activate fluorescence in the cell within 4 min of guanidine treatment, which is at least 15 times faster than a reporter derived from the same riboswitch, and this rapid sensing activity is maintained for up to 1.6 weeks. Together, this study describes the design of two new biosensor topologies and showcases the advantages of RBF biosensors for monitoring dynamic processes in cell biology, biotechnology, and synthetic biology.

Breaking Riboswitch Speed Limits by Systematic Mutation and Orthogonal Base Pair Incorporation

“…We recently reported an RBF biosensor for the metabolite and environmental pollutant, guanidine, that gets activated within a few minutes of analyte addition both in vitro and in vivo (Figure 1A, Figure S1). [3] In contrast, the fluorogenic aptamer by itself, Spinach2, displays much faster fluorescence turn-on (Figure S2A) and was previously shown to be pre-organized for dye binding. [4] The slower turn-on response of the biosensor is due to its fluorogenic mechanism, which requires that the riboswitchligand interaction control folding of the fluorogenic aptamer to affect dye binding.…”

“…[2] While researchers mainly have focused on selectivity and sensitivity in biosensor design, recently we have been interested in understanding and overcoming limitations in RNA-based biosensor kinetics. [3] Access to faster biosensors would expand their capabilities for both real-time diagnostic and molecular imaging applications.…”

An intelligent synthetic bacterium with sound-integrated ability for chronological toxicant detection, degradation, and lethality

“…Regarding detection, whole-cell biosensors have attracted attention because of their low cost, high selectivity, and ease of manufacturing (2). Nucleic acid-and protein-based biosensors are the two most common types that are able to undergo conformational alterations upon input ligand binding to regulate the expression of output signals (3), such as the guanidine-bound S. acidophilus guanidine-I riboswitch, ArsR for arsenic detection, MerR for mercury detection, and DmpR for detecting organophosphate pesticides containing phenolic groups (4)(5)(6). Regarding degradation, analysis of the catabolic pathways in natural strains facilitates the migration of functional genes to artificial cells that do not possess efficient or complete degradation abilities (5,7).…”

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

“…3,4 While researchers mainly have focused on selectivity and sensitivity in biosensor design, recently, we have been interested in understanding and overcoming limitations in RNA-based biosensor response time. 5 Access to faster biosensors would expand their capabilities for both real-time diagnostic and molecular imaging applications.…”

“…Riboswitches are natural structure-switching RNAs that recognize small molecule ligands with high specificity and affinity and can be fused to fluorogenic aptamers to make RNA-based fluorescent (RBF) biosensors. , While researchers mainly have focused on selectivity and sensitivity in biosensor design, recently, we have been interested in understanding and overcoming limitations in RNA-based biosensor response time . Access to faster biosensors would expand their capabilities for both real-time diagnostic and molecular imaging applications.…”