Abstract
G-quadruplex (G4) DNAzymes are guanine-rich oligonucleotides with intrinsic peroxidase-mimicking activity upon complexation with hemin, offering a promising alternative to protein-based enzymes in biosensing. However, their relatively low catalytic efficiency limits practical applications. Here, we present a structure-guided redesign of the high-activity aptamer [B7]-3-0 by incorporating strategic flanking and looping nucleobase modifications. Introduction of adenine and thymine–cytosine elements at the 3′ end led to up to 4-fold enhancements in reaction extent and a 3-fold increase in initial velocity under moderate hydrogen peroxide conditions (0.425 mM). Remarkably, the modified B730 variants retained activity at elevated H₂O₂ concentrations (4.25 mM), achieving up to 8-fold catalytic enhancement and outperforming high-activity DNAzymes including AS1411 and CatG4. These redesigned DNAzymes demonstrated improved peroxidase activity and resistance to oxidative inactivation, addressing a major limitation of both natural and artificial peroxidases. Our findings establish flanking and loop engineering as a cost-effective and broadly applicable strategy for optimizing G4 DNAzymes and underscore their potential in the development of next-generation biosensors.
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Funding
This work was supported by the UKRI/BBSRC grant BB/X012085/1 to F.J.O., and Liverpool John Moores University institutional support to R.I.A. Funding for open access charge: UKRI/BBSRC.
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Conceptualisation: Raphael I. Adeoye and Femi J. Olorunniji; Methodology: Raphael I. Adeoye, Francesca Giuntini and Femi J. Olorunniji; Investigation: Raphael I. Adeoye, Nikhil Babbudas and Matthew Birchenough; Writing—Original Draft: Raphael I. Adeoye, Nikhil Babbudas, Matty Birchenough, and Femi J. Olorunniji; Writing—Review and Editing: Raphael I. Adeoye, Francesca Giuntini and Femi J. Olorunniji; Formal Analysis, Visualisation and Data Curation: Raphael I. Adeoye and Femi J. Olorunniji; Supervision and Project Administration, Femi J. Olorunniji; Funding Acquisition: Raphael I. Adeoye and Femi J. Olorunniji. All authors have read and agreed to the published version of the manuscript.
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Adeoye, R.I., Babbudas, N., Birchenough, M. et al. Rational redesign of high-activity G-quadruplex DNAzyme through flanking and looping of nucleobases. Sci Rep (2026). https://doi.org/10.1038/s41598-026-35686-0
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DOI: https://doi.org/10.1038/s41598-026-35686-0
