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 katalytická * chemie   metabolismus   MeSH
• fosforylace MeSH
• kolorimetrie * metody   MeSH
• konformace nukleové kyseliny MeSH
• oligonukleotidy chemie   MeSH
• substrátová specifita MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA katalytická * MeSH
• oligonukleotidy 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 katalytická * chemie   metabolismus   MeSH
• fluorescence MeSH
• fluorescenční barviva * chemie   MeSH
• kumariny chemie   MeSH
• lidé MeSH
• rychlé screeningové testy * metody   MeSH
• SARS-CoV-2 enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA katalytická * MeSH
• fluorescenční barviva * MeSH
• kumariny 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.

• Klíčová slova
• aptamer, deoxyribozyme, in vitro selection, ribozyme, supernova deoxyribozyme,
• MeSH
• DNA katalytická * metabolismus   MeSH
• DNA MeSH
• konformace nukleové kyseliny MeSH
• nukleotidy MeSH
• nukleové kyseliny * MeSH
• RNA katalytická * metabolismus   MeSH
• RNA genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• DNA katalytická * MeSH
• DNA MeSH
• nukleotidy MeSH
• nukleové kyseliny * MeSH
• RNA katalytická * MeSH
• RNA MeSH