Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Physical Sciences, Biochemical Research Methods, Chemistry, Analytical, Biochemistry & Molecular Biology, Chemistry, DNAzyme, MNAzyme, Room temperature, 10-23 core, IN-VITRO SELECTION, RNA-CLEAVING DNAZYMES, CATALYTIC DNA, AMPLIFICATION, RIBOZYME, NANODEVICES, MICRORNA, ION, PH, 10–23 core, Catalysis, DNA, DNA, Catalytic, DNA, Single-Stranded, Protein Engineering, Reproducibility of Results, Substrate Specificity, Temperature, Thermodynamics, 03 Chemical Sciences, 06 Biological Sciences, 09 Engineering, Analytical Chemistry, 31 Biological sciences, 34 Chemical sciences, 40 Engineering

Abstract:

DNA- and MNAzymes are nucleic acid-based enzymes (NAzymes), which infiltrated the otherwise protein-rich field of enzymology three decades ago. The 10-23 core NAzymes are one of the most widely used and well-characterized NAzymes, but often require elevated working temperatures or additional complex modifications for implementation at standard room temperatures. Here, we present a generally applicable method, based on thermodynamic principles governing hybridization, to re-engineer the existing 10-23 core NAzymes for use at 23 °C. To establish this, we first assessed the activity of conventional NAzymes in the presence of cleavable and non-cleavable substrate at 23 °C as well as over a temperature gradient. These tests pointed towards a non-catalytic mechanism of signal generation at 23 °C, suggesting that conventional NAzymes are not suited for use at this temperature. Following this, several novel NAzyme-substrate complexes were re-engineered from the conventional ones and screened for their performance at 23 °C. The complex with substrate and substrate-binding arms of the NAzymes shortened by four nucleotides on each terminus demonstrated efficient catalytic activity at 23 °C. This has been further validated over a dilution of enzymes or enzyme components, revealing their superior performance at 23 °C compared to the conventional 10-23 core NAzymes at their standard operating temperature of 55 °C. Finally, the proposed approach was applied to successfully re-engineer three other new MNAzymes for activity at 23 °C. As such, these re-engineered NAzymes present a remarkable addition to the field by further widening the diverse repertoire of NAzyme applications.