Engineering and characterization of fluorogenic glycine riboswitches

Nucleic Acids Res         online article

A set of 12 fluorogenic glycine riboswitches with dif- ferent thermodynamic and kinetic response proper- ties was engineered. For the design of functional ri- boswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, trans- mitter and actuator part together. For the RNA sen- sor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluoro- genic aptamer Spinach, respectively. To achieve op- timal signal transduction from the sensor to the ac- tuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integra- tion chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch li- brary. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570  M. Furthermore, the kinetic glycine binding (kon), and dissociation (koff) rates, and corre- sponding energy barriers of the 10 strongest fluores- cence response riboswitches were determined with the same chip platform. kon and koff were in the order of 10−3 s−1 and 10−2 s−1 , respectively. Conclusively, we demonstrate that systematic screening of syn- thetic 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.