title: Systematic reconstruction of binding and stability landscapes of the fluorogenic aptamer spinach.
abstract: Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new generation of in vitro and in vivo sensors for bioanalytics. With the resolved crystal structure of the RNA/small molecule complex, the engineering map becomes available, but comprehensive information regarding the thermodynamic profile of the molecule is missing. Here, we reconstructed the full thermodynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a systematic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermodynamic profile with single base resolution was used to engineer an improved fluorogenic spinach generation via a directed rather than evolutional approach.
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: David
authors.initials: D
authors.lastname: Fuchs
authors.affiliation: Centre for Biological Signalling Studies - BIOSS, University of Freiburg, Schänzlestrasse 18, 79104 Freiburg, Germany Faculty of Biology, University of Freiburg, Schänzlestrasse 18, 79104 Freiburg, Germany.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Weber
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* / Q000235:genetics; D001593:Benzyl Compounds / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D048288:Imidazolines / Q000737:chemistry*; D056656:Lab-On-A-Chip Devices; D017354:Point Mutation; D013816:Thermodynamics
chemical_list: C560120:3,5-difluoro-4-hydroxybenzylidene imidazolinone; D052157:Aptamers, Nucleotide; D001593:Benzyl Compounds; D005456:Fluorescent Dyes; D048288:Imidazolines