Creation and Characterization of a Fluorescence Signalling DNA Enzyme

Abstract

𝘐𝘯 𝘷𝘪𝘵𝘳𝘰 selection has been widely used to isolate single-stranded DNA molecules from large random-sequence pools that are able to perform a desired catalytic reaction, or bind to a target molecule. Using this technique, we have created a DNA enzyme, named DET22-18, which has a uniquely linked chemical catalysis/real-time signaling capability. It is a true enzyme with a 𝘬cₐₜ of~7 min⁻¹ -the second fastest rate ever reported for a DNA enzyme. The DNA enzyme cleaves a substrate containing a single ribonucleotide linkage embedded in a DNA chain and sandwiched between a fluorophore-labeled deoxyribonucleotide and a quencher-modified deoxyribonucleotide. The ability of DET22-18 to generate a large fluorescence signal provides a useful tool to engineer potential allosteric deoxyribozyme biosensors for real-time detection of important biological targets. To provide a proof of concept that a reporter system can be built from the above signaling DNA enzyme, the cis-acting version of this enzyme, DEC22-18A, was engineered into an allosterically regulated deoxyribozyme biosensor that can report ATP. A preliminary investigation was also conducted to determine a possible secondary structure of the DNA enzyme. This study lays a foundation for pursuing novel signaling DNA enzymes for biological detection directed applications.