Comparative Analysis of G-Quadruplex DNAzyme Scaffolds and Split Modes for Programmable Biosensing

Osalaye, DS, Adeoye, RI, Malomo, SO and Olorunniji, FJ ORCID: 0000-0001-9389-2981 (2025) Comparative Analysis of G-Quadruplex DNAzyme Scaffolds and Split Modes for Programmable Biosensing. Catalysts, 16 (1). pp. 1-15. ISSN 2073-4344

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Abstract

G-quadruplex (G4) DNAzymes, guanine-rich sequences that fold into four-stranded structures and bind hemin to mimic peroxidase activity, are widely used in biosensing. Split G4 DNAzymes offer conditional activation upon target recognition, enabling high specificity and modularity. However, achieving low OFF-state leakage remains a major challenge. Here, we systematically characterized four representative G4 scaffolds, C-myc, Bcl2, PS5.M, and C-kit, under standardized ABTS/H2O2 conditions to assess their kinetic properties and suitability for split designs. C-myc exhibited the highest sustained activity and near-linear concentration dependence, making it ideal for quantitative sensing, while Bcl2 showed durable catalysis suited for extended read windows. C-kit produced rapid bursts with early plateaus, favoring binary outputs, and PS5.M initiated quickly but inactivated rapidly, suggesting potential application of systems requiring fast response. Split-mode analysis revealed that symmetric 2:2 partitions often retained significant activity, whereas asymmetric 3:1 splits reduced but did not eliminate leakage. Among the four G4 DNAzymes, PS5.M demonstrated the most promising OFF-state suppression. Design strategies to minimize leakage including non-classical splits, loop/flank edits, and template-assisted assembly could be used to optimize biosensor functionalities. These findings identify essential factors critical for designing robust split DNAzyme biosensors, advancing applications in diagnostics and molecular logic gates.

Item Type:	Article
Uncontrolled Keywords:	34 Chemical Sciences; 3406 Physical Chemistry; 40 Engineering; 4004 Chemical Engineering; 4018 Nanotechnology; Bioengineering; 0306 Physical Chemistry (incl. Structural); 3406 Physical chemistry; 4004 Chemical engineering; 4018 Nanotechnology
Subjects:	Q Science > Q Science (General) Q Science > QR Microbiology
Divisions:	Pharmacy and Biomolecular Sciences
Publisher:	MDPI AG
Date of acceptance:	26 December 2025
Date of first compliant Open Access:	6 February 2026
Date Deposited:	06 Feb 2026 08:26
Last Modified:	06 Feb 2026 08:26
DOI or ID number:	10.3390/catal16010027
URI:	https://researchonline.ljmu.ac.uk/id/eprint/28050

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