Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades

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.

Thiolate DNAzymes on Gold Nanoparticles for Isothermal Amplification and Detection of Mesothelioma-derived Exosomal PD-L1 mRNA

“…Moreover, when the substrate components within the Subzyme were cleaved by the initiating DNAzyme, the surface-bound 8–17 DNAzymes were released and could successfully cleave their corresponding substrate-FQs, indicating that their catalytic activity was restored (Figure A,B). The results are comparable to our previous work using magnetic microparticles via biotin–streptavidin attachment chemistry. , …”

“…Though the experiments in this study were performed isothermally using a qPCR instrument for fluorescence detection, qPCR equipment is not necessary, as simple and compact devices can be engineered for temperature management and fluorescence detection. Moreover, the cross-catalytic cascade platform could be further explored using AuNPs and dithiol-modified Subzymes to further improve the LoD. Further development, clinical translation, and commercialization will lead to applications in a broad range of fields, including forensic, medical, agricultural, environmental, and veterinary.…”

“…To build on their bioassay capabilities, DNAzymes have been used to construct signal amplification cascades to further improve the limit of detection, where target concentrations are low. − In a recent study, DNAzyme structures, referred to as Subzymes, were attached to magnetic microparticles for regulating DNAzyme activity in response to nucleic acid targets . In another study, when Subzymes combined with PlexZymes, also known as MNAzymes, the signal amplification properties of Subzymes have been applied for the detection of nucleic acid targets. However, this system is based on Subzyme attachment to microparticles via biotin–streptavidin bonds, which is less stable under high temperatures .…”

Toward a Home Test for COVID‐19 Diagnosis: DNA Machine for Amplification‐Free SARS‐CoV‐2 Detection in Clinical Samples

“…Amplification of such RNA samples require annealing step requiring heating equipment, which adds to the complexity and the cost of RNA analysis. BiDz probe was reported to be efficient in detecting short DNA analytes, [12][13][14][15] but is less successful in reporting the presence of long viral RNA as was found in this study (Figure 2A). Here, we proposed and tested the strategy for designing a sensor that can efficiently recognize natural viral RNA.…”

“…[12] BiDz can reach LOD down to 1–10 pM, [13] which is, to the best of our knowledge, the lowest among protein enzyme‐free testing systems that do not use amplification cascades. [14] BiDz consists of two DNA strands, Dz a and Dz b in Figure 1 A which hybridize to the analyzed nucleic acid and form Dz catalytic core followed by the cleavage of fluorophore and a quencher‐labelled reporter substrate (F sub in Figure 1 ). [12] One advantage of BiDz is its ability to amplify the signal due to cleaving multiple F sub molecules.…”