Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades
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-catal...
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doaj-636fc51726f0436396210195861970742020-11-25T02:04:12ZengMDPI AGMolecules1420-30492020-04-01251755175510.3390/molecules25071755Sensitive Detection of Nucleic Acids Using Subzyme Feedback CascadesNicole Hasick0Andrea Lawrence1Radhika Ramadas2Alison Todd3SpeeDx, Pty Ltd., Eveleigh, NSW 2015, AustraliaSpeeDx, Pty Ltd., Eveleigh, NSW 2015, AustraliaSpeeDx, Pty Ltd., Eveleigh, NSW 2015, AustraliaSpeeDx, Pty Ltd., Eveleigh, NSW 2015, AustraliaThe 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<sup>®</sup> 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 <i>Chlamydia trachomatis</i> 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.https://www.mdpi.com/1420-3049/25/7/1755catalytic DNAdeoxyribozymeDNAzymePlexZymesignal amplificationisothermal |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Nicole Hasick Andrea Lawrence Radhika Ramadas Alison Todd |
spellingShingle |
Nicole Hasick Andrea Lawrence Radhika Ramadas Alison Todd Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades Molecules catalytic DNA deoxyribozyme DNAzyme PlexZyme signal amplification isothermal |
author_facet |
Nicole Hasick Andrea Lawrence Radhika Ramadas Alison Todd |
author_sort |
Nicole Hasick |
title |
Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades |
title_short |
Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades |
title_full |
Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades |
title_fullStr |
Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades |
title_full_unstemmed |
Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades |
title_sort |
sensitive detection of nucleic acids using subzyme feedback cascades |
publisher |
MDPI AG |
series |
Molecules |
issn |
1420-3049 |
publishDate |
2020-04-01 |
description |
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<sup>®</sup> 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 <i>Chlamydia trachomatis</i> 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. |
topic |
catalytic DNA deoxyribozyme DNAzyme PlexZyme signal amplification isothermal |
url |
https://www.mdpi.com/1420-3049/25/7/1755 |
work_keys_str_mv |
AT nicolehasick sensitivedetectionofnucleicacidsusingsubzymefeedbackcascades AT andrealawrence sensitivedetectionofnucleicacidsusingsubzymefeedbackcascades AT radhikaramadas sensitivedetectionofnucleicacidsusingsubzymefeedbackcascades AT alisontodd sensitivedetectionofnucleicacidsusingsubzymefeedbackcascades |
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1724943872570687488 |