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Open AccessArticle

Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades

1
SpeeDx, Pty Ltd., Eveleigh, NSW 2015, Australia
2
Biotechnology and Biomolecular Sciences, The University of New South Wales, Kensington, NSW 2052, Australia
*
Author to whom correspondence should be addressed.
Academic Editors: Michael Smietana, Stellios Arseniyadis and Sabine Müller
Molecules 2020, 25(7), 1755; https://doi.org/10.3390/molecules25071755
Received: 16 March 2020 / Revised: 3 April 2020 / Accepted: 8 April 2020 / Published: 10 April 2020
(This article belongs to the Special Issue Advances in Catalytic DNA)
The development of Subzymes demonstrates how the catalytic activity of DNAzymes can be controlled for detecting nucleic acids; however, Subzymes alone lack the sensitivity required to detect low target concentrations. To improve sensitivity, we developed a feedback system using a pair of cross-catalytic Subzymes. These were individually tethered to microparticles (MP) and separated by a porous membrane rendering them unable to interact. In the presence of a target, active PlexZymes® cleave a first Subzyme, which separates a first DNAzyme from its MP, allowing the DNAzyme to migrate through the membrane, where it can cleave a second Subzyme. This releases a second DNAzyme which can now migrate through the membrane and cleave more of the first Subzyme, thus initiating a cross-catalytic cascade. Activated DNAzymes can additionally cleave fluorescent substrates, generating a signal, and thereby, indicating the presence of the target. The method detected 1 fM of DNA homologous to the ompA gene of Chlamydia trachomatis within 30 min, demonstrating a 10,000-fold increase in sensitivity over PlexZyme detection alone. The Subzyme cascade is universal and can be triggered by any target by modifying the target sensing arms of the PlexZymes. Further, it is isothermal, protein-enzyme-free and shows great potential for rapid and affordable biomarker detection. View Full-Text
Keywords: catalytic DNA; deoxyribozyme; DNAzyme; PlexZyme; signal amplification; isothermal; catalytic DNA; feedback cascade; point of care catalytic DNA; deoxyribozyme; DNAzyme; PlexZyme; signal amplification; isothermal; catalytic DNA; feedback cascade; point of care
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MDPI and ACS Style

Hasick, N.; Lawrence, A.; Ramadas, R.; Todd, A. Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades. Molecules 2020, 25, 1755.

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