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Ketterer, S.* ; Gladis, L.* ; Kozica, A.* ; Meier, M.

Engineering and characterization of fluorogenic glycine riboswitches.

Nucleic Acids Res. 44, 5983-5992 (2016)
DOI PMC
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Open Access Gold as soon as Publ. Version/Full Text is submitted to ZB.
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.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
ISSN (print) / ISBN 0305-1048
e-ISSN 1362-4962
Journal Nucleic Acids Research
Quellenangaben Volume: 44, Issue: 12, Pages: 5983-5992 Article Number: , Supplement: ,
Publisher Oxford University Press
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Helmholtz Pioneer Campus (HPC)