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

For many decades it was thought that information storage and information transfer were the main functions of nucleic acids. However, artificial evolution experiments have shown that the functional potential of DNA and RNA is much greater. Here I provide an overview of this technique and highlight recent advances which have increased its potency. I also describe how artificial evolution has been used to identify nucleic acids with extreme functions. These include deoxyribozymes that generate unusual products such as light, tiny motifs made up of fewer than ten nucleotides, ribozymes that catalyze complex reactions such as RNA polymerization, information-rich sequences that encode overlapping ribozymes, motifs that catalyze reactions at rates too fast to be followed by manual pipetting, and functional nucleic acids which are active in extreme conditions. Such motifs highlight the limits of our knowledge and provide clues about as of yet undiscovered functions of DNA and RNA.

• Keywords
• aptamer, deoxyribozyme, in vitro selection, ribozyme, supernova deoxyribozyme,
• MeSH
• DNA, Catalytic * metabolism   MeSH
• DNA MeSH
• Nucleic Acid Conformation MeSH
• Nucleotides MeSH
• Nucleic Acids * MeSH
• RNA, Catalytic * metabolism   MeSH
• RNA genetics   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA, Catalytic * MeSH
• DNA MeSH
• Nucleotides MeSH
• Nucleic Acids * MeSH
• RNA, Catalytic * MeSH
• RNA MeSH

Artificial evolution experiments typically use libraries of ∼1015 sequences and require multiple rounds of selection to identify rare variants with a desired activity. Based on the simple structures of some aptamers and nucleic acid enzymes, we hypothesized that functional motifs could be isolated from significantly smaller libraries in a single round of selection followed by high-throughput sequencing. To test this idea, we investigated the catalytic potential of DNA architectures in which twelve or fifteen randomized positions were embedded in a scaffold present in all library members. After incubating in either the presence or absence of lead (which promotes the nonenzymatic cleavage of RNA), library members that cleaved themselves at an RNA linkage were purified by PAGE and characterized by high-throughput sequencing. These selections yielded deoxyribozymes with activities 8- to 30-fold lower than those previously isolated under similar conditions from libraries containing 1014 different sequences, indicating that the disadvantage of using a less diverse pool can be surprisingly small. It was also possible to elucidate the sequence requirements and secondary structures of deoxyribozymes without performing additional experiments. Due to its relative simplicity, we anticipate that this approach will accelerate the discovery of new catalytic DNA and RNA motifs.

• MeSH
• DNA, Catalytic chemistry   isolation & purification   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Nucleotide Motifs MeSH
• RNA Cleavage MeSH
• Substrate Specificity MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Catalytic MeSH