Colorimetric assays in which the color of a solution changes in the presence of an input provide a simple and inexpensive way to monitor experimental readouts. In this study we used in vitro selection to identify a self-phosphorylating kinase deoxyribozyme that produces a colorimetric signal by converting the colorless substrate pNPP into the yellow product pNP. The minimized catalytic core, sequence requirements, secondary structure, and buffer requirements of this deoxyribozyme, which we named Apollon, were characterized using a variety of techniques including reselection experiments, high-throughput sequencing, comparative analysis, biochemical activity assays, and NMR. A bimolecular version of Apollon catalyzed multiple turnover phosphorylation and amplified the colorimetric signal. Engineered versions of Apollon could detect oligonucleotides with specific sequences as well as several different types of nucleases in homogenous assays that can be performed in a single tube without the need for washes or purifications. We anticipate that Apollon will be particularly useful to reduce costs in high-throughput screens and for applications in which specialized equipment is not available.

• MeSH
• DNA, Catalytic * chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Colorimetry * methods   MeSH
• Nucleic Acid Conformation MeSH
• Oligonucleotides chemistry   MeSH
• Substrate Specificity MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Catalytic * MeSH
• Oligonucleotides MeSH

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 homogenous 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.

• MeSH
• DNA, Catalytic * chemistry   metabolism   MeSH
• Fluorescence MeSH
• Fluorescent Dyes * chemistry   MeSH
• Coumarins chemistry   MeSH
• Humans MeSH
• High-Throughput Screening Assays * methods   MeSH
• SARS-CoV-2 enzymology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Catalytic * MeSH
• Fluorescent Dyes * MeSH
• Coumarins MeSH