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.
CITATION STYLE
Ven, K., Safdar, S., Dillen, A., Lammertyn, J., & Spasic, D. (2019). Re-engineering 10–23 core DNA- and MNAzymes for applications at standard room temperature. Analytical and Bioanalytical Chemistry, 411(1), 205–215. https://doi.org/10.1007/s00216-018-1429-4
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