Peroxidase DNAzymes are single-stranded, stable G-quadruplexes structures that exhibit catalytic activity with cofactor hemin. This class of DNAzymes offers several advantages over traditional protein and RNA catalysts, including thermal stability, resistance to hydrolysis, and easy of synthesis in the laboratory. However, their use in medicine, biology, and chemistry is limited due to their low catalytic rates. Selecting and evolving for higher catalytic rates has been challenging due to limitations in selection methodology which generally use affinity as the selection pressure instead of kinetics. We previously evolved a new peroxidase DNAzyme (mSBDZ-X-3) through a directed evolution method, which was subsequently used for proximity labelling in a proteomic experiment in cell culture. Herein, we present a detailed protocol for this function-based laboratory evolution method to evolve peroxidase DNAzymes for future laboratory implementation. This approach is based on capturing self-biotinylated DNA, which is catalyzed by intrinsic peroxidase activity to select for DNAzyme molecules. The selection method uses fluorescence-based real-time monitoring of the DNA pools, allowing for the enrichment of catalytic activity and capture of catalytic DNA across evolutionary selection rounds. The evolved mSBDZ-X-3 DNAzyme attributes parallel G-quadruplex structure and demonstrates better catalytic properties than DNAzyme variants evolved previously. The influence of critical reaction parameters is outlined. This protocol enables discovery of improved peroxidase DNAzyme/RNAzyme variants from natural or chemical-modified nucleotide libraries. The approach could be applicable for the selection of catalytic activities in a variety of directed molecular evolution contexts.
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http://dx.doi.org/10.1093/biomethods/bpae088 | DOI Listing |
Biol Methods Protoc
December 2024
School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, P.R. China.
Peroxidase DNAzymes are single-stranded, stable G-quadruplexes structures that exhibit catalytic activity with cofactor hemin. This class of DNAzymes offers several advantages over traditional protein and RNA catalysts, including thermal stability, resistance to hydrolysis, and easy of synthesis in the laboratory. However, their use in medicine, biology, and chemistry is limited due to their low catalytic rates.
View Article and Find Full Text PDFSmall
January 2025
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, China.
Emerging evidence demonstrates that inducing ferroptosis, a nonapoptotic programmed cell death mode, holds significant potential for tumor treatment. However, current ferroptosis strategies utilizing exogenous Fenton-type heavy metal species or introducing glutathione (GSH)/glutathione peroxidase 4 (GPX4) suppressants are hampered by latent adverse effects toward organisms, while utilizing endogenous iron may cause undesirable tumor angiogenesis through specific signaling pathways. Here, a ferric ion (Fe)-responsive and DNAzyme-delivered coordination nanosystem (ZDD) is developed to achieve a novel scheme of synergistic tumor-specific ferroptosis and gene therapy, which modulates and harnesses the endogenous iron in tumors for inducing ferroptosis while intercepting tumor angiogenesis to enhance therapeutic efficacy.
View Article and Find Full Text PDFBiosensors (Basel)
January 2025
School of Pharmacy & Biomolecular Sciences, Faculty of Health, Innovation, Technology and Science, Liverpool John Moores University, Liverpool L3 3AF, UK.
G-quadruplex (G4) DNAzymes with peroxidase activities hold potential for applications in biosensing. While these nanozymes are easy to assemble, they are not as efficient as natural peroxidase enzymes. Several approaches are being used to better understand the structural basis of their reaction mechanisms, with a view to designing constructs with improved catalytic activities.
View Article and Find Full Text PDFBiosens Bioelectron
January 2025
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, PR China; University of Chinese Academy of Sciences, Beijing, 100049, PR China. Electronic address:
The development of point-of-care testing (POCT) methods is highly desirable in molecular detection, as they enable disease diagnosis and biomarker monitoring on-site or at home. Repurposing existing POCT devices to detect diverse biomarkers is an economical way to develop new devices for POCT use. Personal glucose meter (PGM) is one of the most used off-the-shelf POCT devices that has been reused to detect non-glucose targets.
View Article and Find Full Text PDFTalanta
January 2025
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Materials Science, Hebei University, Baoding 071002, Hebei Province, PR China. Electronic address:
MicroRNAs (miRNAs) serve as potential biomarkers for many diseases such as cancer, neurodegenerative diseases and cardiovascular conditions. The portable and accurate detection of miRNA is of great significance for the early diagnosis, treatment optimization and prognostic evaluation of diseases. Herein, a photothermal/visual dual-mode assay for let-7a is developed utilizing oxidized 3, 3', 5, 5' - tetramethylbenzidine (oxTMB) as signal reporter.
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