title: 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 (k(on)), and dissociation (k(off)) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. k(on) and k(off) were in the order of 10(-3)s(-1) and 10(-2)s(-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.
authors.affiliation: Microfluidic and Biological Engineering, Department of Microsystems Engineering-IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany Centre for Biological Signalling Studies-BIOSS, University of Freiburg, Schänzlestrasse 18, 79104 Freiburg, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Ketterer
authors.affiliation: Microfluidic and Biological Engineering, Department of Microsystems Engineering-IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany Centre for Biological Signalling Studies-BIOSS, University of Freiburg, Schänzlestrasse 18, 79104 Freiburg, Germany.
authors.forename: Lukas
authors.initials: L
authors.lastname: Gladis
authors.affiliation: Microfluidic and Biological Engineering, Department of Microsystems Engineering-IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany Centre for Biological Signalling Studies-BIOSS, University of Freiburg, Schänzlestrasse 18, 79104 Freiburg, Germany.
authors.forename: Adnan
authors.initials: A
authors.lastname: Kozica
authors.affiliation: Microfluidic and Biological Engineering, Department of Microsystems Engineering-IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany Centre for Biological Signalling Studies-BIOSS, University of Freiburg, Schänzlestrasse 18, 79104 Freiburg, Germany matthias.meier@imtek.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Meier
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D052158:Aptamers, Peptide / Q000138:chemical synthesis / Q000737:chemistry*; D001412:Bacillus subtilis / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D015723:Gene Library; D005818:Genetic Engineering; D005998:Glycine / Q000032:analysis / Q000737:chemistry*; D007700:Kinetics; D056656:Lab-On-A-Chip Devices; D009843:Oligoribonucleotides / Q000737:chemistry; D058928:Riboswitch; D015398:Signal Transduction; D013816:Thermodynamics
chemical_list: D052157:Aptamers, Nucleotide; D052158:Aptamers, Peptide; D005456:Fluorescent Dyes; D009843:Oligoribonucleotides; D058928:Riboswitch; D005998:Glycine