Development of an aptamer-based screening for identification and characterizationof novel RNA therapeutics using switchSENSE® technology.

Abstract

Approximately 85 % of the genes are transcribed into RNA but only 3 % are translated into proteins. This is not reflected in the amount of available drugs, as most of them target proteins which relate to 0.05 % of nucleic acids. Thus, an effort must be made to fill this gap and focus on the transcriptome level of diseases. Non-coding RNAs are especially interesting as they account for the major share of the transcriptome and fulfill important regulatory functions as small interfering (siRNA), transfer-RNA (tRNA) and other unknown ones. Due to their importance, they could be explored as novel drug targets, e.g. to develop alternative antibiotics. A successful example in procaryotes is targeting the synthesis of the highly conserved and essential survival factor flavin mononucleotide (FMN) by its riboswitch which can modulate the expression over structural re-arrangement. Identifying small molecules binding to the aptamer region of these riboswitches to disrupt its synthesis is an innovative approach for developing alternative antibiotics. Different methods are used to identify small molecules, like structural approaches by NMR, thermodynamic changes with ITC or radius of gyration by SAXS. Herein, we developed biochips to overcome pricy sample preparation and consumption, with which we measure binding kinetics of molecules targeting aptamers using switchSENSE® biosensing technology. switchSENSE® is based on electro-switchable DNA-layers immobilized on a gold surface. The 2-color optical detection system allows on-chip FRET experiments to monitor not only binding, but also structural re-arrangements. As proof of concept we studied the binding-induced folding of the cocaine-binding aptamer with different small molecules. This tool delivers fast results on small molecule screenings for detailed binding kinetics and binding-induced folding kinetics which can improve the search of novel drugs at the transcriptome level.