Abstract
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.
Highlights
Catalytic nucleic acids (CNAs) such as DNAzymes, ribozymes and PlexZymes® contain the elements required for biosensing, namely, molecular recognition and transducer components
The schema illustrates the application of a Porous Membrane Bags (PMBs), which encloses and physically separates certain assay components from others, such that it allows the passage of certain substances through it while preventing that of others
A pair of Subzymes that contain complementary substrate and DNAzyme components are attached to microparticles (MPs) which are too large to pass though the PMB
Summary
Catalytic nucleic acids (CNAs) such as DNAzymes, ribozymes and PlexZymes® contain the elements required for biosensing, namely, molecular recognition and transducer components. As such, they are effective tools for constructing nucleic acid biosensors. We reported the development of novel CNA structures called Subzymes [1]. Subzymes contain both a universal substrate and a CNA enzyme, and can serve as molecular intermediaries for linking target recognition with signal output. We demonstrated that the activity of certain CNAs can be inhibited by tethering Subzymes to microparticles (Subzyme-MP), and that this inhibition can be reversed by cleaving a Subzyme’s internal substrate and releasing the CNA in response to the presence of target DNA.
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