Engineering and characterization of fluorogenic glycine riboswitches

Abstract

A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch library. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570 μM. Furthermore, the kinetic glycine binding (kon), and dissociation (koff) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. kon and koff were in the order of 10−3s−1 and 10−2s−1, respectively. Conclusively, we demonstrate that systematic screening of synthetic and natural linked RNA parts with microfluidic chip technology is an effective approach to rapidly generate fluorogenic metabolite riboswitches with a broad range of biophysical response properties.

Design of Artificial Riboswitches as Biosensors

“…Similar to GFP, Spinach variants with different colors could be generated. The Spinach aptamer was successfully used to monitor metabolite binding to natural riboswitch aptamers [ 54 , 55 , 56 , 57 , 58 ]. A second fluorescent DFHBI-binding aptamer is Broccoli, also isolated by SELEX, but optimized for in vivo applications by cellular screening [ 59 ].…”

Sensitive and specific detection of ligands using engineered riboswitches

“…Although much work has focused on using riboswitches as biosensors in vivo, less effort has been exerted toward using riboswitches to detect ligands in vitro. One highly successful approach for making sensors for both in vitro and in vivo detection of ligands has been to fuse various aptamer domains via a short "communication module" to a fluorescent aptamer such as "Spinach" (Litke et al, 2016;Paige et al, 2011;Nakayama et al, 2012;Kellenberger et al, 2013;Bhadra and Ellington, 2014;Kellenberger et al, 2015;Ketterer et al, 2016;Bose et al, 2016). The fusions are designed such that ligand binding to the aptamer domain allows the fluorescent aptamer to fold into its active conformation.…”

Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More

“…Finally, also microarrays were used for in vitro functional screening of aptamers [44][45][46]. Upon DNA spotting onto a surface, genes are transcribed and captured either onto a functionalized coverslip [46] or into a dedicated micro-chamber [44].…”