A fluorescent deoxyribozyme for high-throughput screening

M. Volek1-2, J. Kurfürst1,3, M. Drexler1, M. Svoboda1, P. Srb1, V. Veverka1,4, E.A. Curtis1

1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 166 10, Prague, Czech Republic

2Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, 128 44, Prague, Czech Republic

3Department of Informatics and Chemistry, University of Chemistry and Technology, 166 28, Prague, Czech Republic

2Department of Cell Biology, Faculty of Science, Charles University in Prague, 128 44, Prague, Czech Republic

curtis@uochb.cas.cz

Fluorescence facilitates the detection, visualization, and tracking of molecules with high sensitivity and specificity. A functional DNA molecule that generates a robust fluorescent signal would offer significant advantages for many applications compared to intrinsically fluorescent proteins, which are expensive and labor intensive to synthesize, and fluorescent RNA aptamers, which are unstable under most conditions. Here we describe a novel deoxyriboyzme that rapidly and efficiently generates a stable fluorescent product using a readily available coumarin substrate. An engineered version can detect picomolar concentrations of ribonucleases in a simple homogeneous assay, and was used to rapidly identify novel inhibitors of the SARS-CoV-2 ribonuclease Nsp15 in a high-throughput screen. Our work adds an important new component to the toolkit of functional DNA parts, and also demonstrates how catalytic DNA motifs can be used to solve real-world problems.