{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T08:16:15Z","timestamp":1773044175537,"version":"3.50.1"},"reference-count":23,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2020,4,10]],"date-time":"2020-04-10T00:00:00Z","timestamp":1586476800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>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\u00ae 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.<\/jats:p>","DOI":"10.3390\/molecules25071755","type":"journal-article","created":{"date-parts":[[2020,4,14]],"date-time":"2020-04-14T03:10:01Z","timestamp":1586833801000},"page":"1755","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades"],"prefix":"10.3390","volume":"25","author":[{"given":"Nicole","family":"Hasick","sequence":"first","affiliation":[{"name":"SpeeDx, Pty Ltd., Eveleigh, NSW 2015, Australia"},{"name":"Biotechnology and Biomolecular Sciences, The University of New South Wales, Kensington, NSW 2052, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5863-8985","authenticated-orcid":false,"given":"Andrea","family":"Lawrence","sequence":"additional","affiliation":[{"name":"SpeeDx, Pty Ltd., Eveleigh, NSW 2015, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Radhika","family":"Ramadas","sequence":"additional","affiliation":[{"name":"SpeeDx, Pty Ltd., Eveleigh, NSW 2015, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alison","family":"Todd","sequence":"additional","affiliation":[{"name":"SpeeDx, Pty Ltd., Eveleigh, NSW 2015, Australia"},{"name":"Biotechnology and Biomolecular Sciences, The University of New South Wales, Kensington, NSW 2052, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,4,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"126704","DOI":"10.1016\/j.snb.2019.126704","article-title":"Subzymes: Regulating DNAzymes for point of care nucleic acid sensing","volume":"297","author":"Hasick","year":"2019","journal-title":"Sensors Actuators B Chem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2881","DOI":"10.1021\/cr400354z","article-title":"From cascaded catalytic nucleic acids to enzyme-DNA nanostructures: Controlling reactivity, sensing, logic operations, and assembly of complex structures","volume":"114","author":"Wang","year":"2014","journal-title":"Chem. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.bios.2015.08.021","article-title":"EzyAmp signal amplification cascade enables isothermal detection of nucleic acid and protein targets","volume":"75","author":"Linardy","year":"2016","journal-title":"Biosens. Bioelectron."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"9106","DOI":"10.1021\/ac501811r","article-title":"V DNA-only cascade: A universal tool for signal amplification, enhancing the detection of target analytes","volume":"86","author":"Bone","year":"2014","journal-title":"Anal. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"7737","DOI":"10.1038\/srep07737","article-title":"Restriction cascade exponential amplification (RCEA) assay with an attomolar detection limit: A novel, highly specific, isothermal alternative to qPCR","volume":"5","author":"Ghindilis","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_6","first-page":"1","article-title":"A DNAzyme feedback amplification strategy for biosensing","volume":"56","author":"Liu","year":"2017","journal-title":"Angew. Chemie Int. Ed."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"4473","DOI":"10.1002\/chem.201705338","article-title":"DNAzyme Feedback Amplification: Relaying Molecular Recognition to Exponential DNA Amplification","volume":"24","author":"Liu","year":"2018","journal-title":"Chem. -A Eur. J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/j.aca.2019.10.051","article-title":"Catalytic hairpin assembly-based double-end DNAzyme cascade-feedback amplification for sensitive fluorescence detection of HIV-1 DNA","volume":"1096","author":"Liu","year":"2020","journal-title":"Anal. Chim. Acta"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1002\/anie.201005246","article-title":"Amplified detection of DNA through an autocatalytic and catabolic DNAzyme-mediated process","volume":"50","author":"Wang","year":"2011","journal-title":"Angew. Chemie-Int. Ed."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1097","DOI":"10.1093\/nar\/gky1245","article-title":"Allosteric DNAzyme-based DNA logic circuit: Operations and dynamic analysis","volume":"47","author":"Zheng","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1038\/nrm1838","article-title":"Cell signalling dynamics in time and space","volume":"7","author":"Kholodenko","year":"2006","journal-title":"Nat Rev Mol Cell Biol"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Yang, F., Liu, Y., Wang, B., Zhou, C., and Zhang, Q. (2019). Constructing controllable logic circuits based on DNAzyme activity. Molecules, 24.","DOI":"10.3390\/molecules24224134"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"11714","DOI":"10.1021\/acs.analchem.5b02654","article-title":"Entropy beacon: A hairpin-free DNA amplification strategy for efficient detection of nucleic acids","volume":"87","author":"Lv","year":"2015","journal-title":"Anal. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5386","DOI":"10.1073\/pnas.1222807110","article-title":"Stacking nonenzymatic circuits for high signal gain","volume":"110","author":"Chen","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Ang, Y.S., Tong, R., and Yung, L.Y.L. (2016). Engineering a robust DNA split proximity circuit with minimized circuit leakage. Nucleic Acids Res., 44.","DOI":"10.1093\/nar\/gkw447"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5389","DOI":"10.1021\/acs.analchem.7b00081","article-title":"Semi-permeable functional DNA-encapsulated nanocapsules as protective bioreactors for biosensing in living cells","volume":"10","author":"Hong","year":"2017","journal-title":"Anal. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"992","DOI":"10.1038\/s41467-017-01072-8","article-title":"Control of enzyme reactions by a reconfigurable DNA nanovault","volume":"8","author":"Grossi","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"15581","DOI":"10.1021\/ja074335t","article-title":"Networking particles over distance using oligonucleotide-based devices","volume":"129","author":"Yashin","year":"2007","journal-title":"J. Am. Chem. Soc."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"6416","DOI":"10.1073\/pnas.1130145100","article-title":"Exponential growth by cross-catalytic cleavage of deoxyribozymogens","volume":"100","author":"Levy","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1016\/j.tibtech.2011.01.007","article-title":"Point-of-care nucleic acid testing for infectious diseases","volume":"29","author":"Niemz","year":"2011","journal-title":"Trends Biotechnol"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1021\/acssensors.6b00691","article-title":"What are clinically relevant levels of cellular and biomolecular analytes?","volume":"2","author":"Kelley","year":"2017","journal-title":"ACS Sensors"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Dirks, J.A.M.C., Van Liere, G.A.F.S., Bogers, S., Dukers-Muijrers, N.H.T.M., Wolffs, P.F.G., and Hoebe, C.J.P.A. (2015). Natural course of Chlamydia trachomatis bacterial load in the time interval between screening and treatment in anogenital samples. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0145693"},{"key":"ref_23","first-page":"1150","article-title":"Laboratory diagnosis of urinary tract infections in adult patients","volume":"38","author":"Wilson","year":"2004","journal-title":"Med. Microbiol."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/25\/7\/1755\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:17:25Z","timestamp":1760174245000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/25\/7\/1755"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,4,10]]},"references-count":23,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2020,4]]}},"alternative-id":["molecules25071755"],"URL":"https:\/\/doi.org\/10.3390\/molecules25071755","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,4,10]]}}}