Systematic reconstruction of binding and stability landscapes of the fluorogenic aptamer spinach

22.09.2015

Ketterer S, Fuchs D, Weber W, Meier M.

Nucleic Acids Res. 2015;43(19):9564-72.

Nucleic Acids Res.            online article

Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imida- zolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new gen- eration of in vitro and in vivo sensors for bioan- alytics. 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 thermo- dynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a system- atic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermody- namic profile with single base resolution was used to engineer an improved fluorogenic spinach gener- ation via a directed rather than evolutional approach.