RNA-cleaving DNAzymes (RCDs) represent a transformative class of functional nucleic acids that catalyze the site-specific cleavage of RNA substrates. Unlike antibodies or aptamers, RCDs combine high catalytic turnover, sequence-specific recognition, and chemical stability. Initially discovered in the mid-1990s for their metal ion-dependent cleavage activity, RCDs have rapidly evolved to target a wide range of clinically relevant analytes, including bacterial strains, protein biomarkers, small metabolites, and viral RNAs.
The 10–23 DNAzyme remains a cornerstone of this class. Dependent on Mg2+ ions, it efficiently cleaves all-RNA substrates at purine-pyrimidine junctions with catalytic constants approaching 10 min-1 under optimal conditions. This unparalleled catalytic rate positions RCDs as ideal components for point-of-care (POC) diagnostic assays.
Fig.1 Overview of selection methods used to generate RNA-cleaving DNAzymes.(Ali M., et al., 2024)
Engineering RCDs: From Library Design to Target-Responsive Cleavage
In Vitro Selection Methodologies
The core of RCD engineering lies in high-stringency in vitro selection, often using systematic evolution of ligands by exponential enrichment (SELEX). Starting with libraries comprising up to 1014 unique sequences, catalytically active species are enriched through positive selection (PS) and counter-selection (CS) cycles.
PAGE-based selection isolates cleaved products via polyacrylamide gel electrophoresis. In contrast, magnetic bead-based selection utilizes biotin-streptavidin interactions to immobilize the library, enabling efficient automation and reduced cycle times. These methods allow fine-tuned selection for target specificity and cleavage kinetics by adjusting pH, buffer conditions, and ion types.
Target Versatility and Specificity
RCDs have demonstrated activation by over 25 different metal ions, bacterial cell extracts, and individual proteins. The RFD-EC1 DNAzyme, for example, cleaves in the presence of E. coli lysate and discriminates against both gram-positive and gram-negative species. On the protein side, EPDz20 M5 is a landmark RCD selected directly against eosinophil peroxidase (EPX), a critical biomarker in asthma.
Mechanisms of Signal Transduction: Catalysis to Quantifiable Output
Cleavage-Based Signal Generation
The hallmark of RCD-based detection is the transesterification reaction at RNA cleavage sites, generating distinct nucleic acid fragments. These products can be engineered with functional tags for signal transduction via:
- Fluorescence dequenching using fluorophore-quencher pairs
- Electrochemical readouts via redox mediators
- Colorimetric shifts using enzyme-linked reactions
- Lateral flow detection using nucleic acid hybridization
Integration with Amplification Schemes
To enhance sensitivity, RCDs are frequently coupled with isothermal amplification techniques. Rolling Circle Amplification (RCA), Loop-Mediated Isothermal Amplification (LAMP), and CRISPR-based methods have been successfully adapted to utilize RCD-cleaved fragments as primers or triggers.
A notable platform, REVEALR, integrates split DNAzymes with reverse transcription-recombinase polymerase amplification (RT-RPA) followed by T7 transcription to detect SARS-CoV-2 RNA at attomolar levels.
Bacterial Pathogen Detection: Differentiation and Clinical Application
Selectivity at the Strain Level
RCDs have shown promise in differentiating bacterial strains in complex matrices. For instance:
- RFD-EC1 targets E. coli with detection limits as low as 500 CFU/mL.
- RFD-CD1 uniquely recognizes the hypervirulent BI/027 strain of C. difficile.
- RFD-SA6T1, although not strain-specific, demonstrates broad detection of both MSSA and MRSA in nasal mucus.
Table 1. Examples of Bacterial-Specific RCDs.
| RCD Identifier |
Target Bacterium |
Specificity |
Detection Limit |
| RFD-EC1 |
E. coli |
Species level |
500 CFU/mL |
| RFD-CD1 |
C. difficile (BI/027) |
Strain-specific |
Not evaluated |
| RFD-KP6 |
Klebsiella pneumoniae |
Species level, multiple strains |
105 CFU/mL |
Integration in Diagnostic Platforms
Paper-based devices for H. pylori (DHp3T4) and S. aureus (RFD-SA6T1) incorporate urease- or gold nanoparticle-tagged DNA strands for visual readout. These devices demonstrate robust performance in matrices like stool and nasal mucus, requiring minimal processing and yielding results within 30 minutes.
Protein and Metabolite Detection: Toward Biomarker-Specific RCDs
Direct Protein Targeting
The recent development of protein-activated DNAzymes marks a pivotal breakthrough. EPDz20 M5, derived via 15 PS rounds and CS steps against other eosinophil proteins, achieves nanomolar detection of EPX in unprocessed sputum.
MORAC (MOlecule Recognition based on Affinity and Catalysis) technology has further advanced the field by allowing simultaneous DNAzyme selection and target protein identification. For example, Dz04 was shown to bind apolipoprotein L6 (APOL6), and Dz41 recognized SARS1 in colon polyp tissues.
Allosteric and Rationally Designed Aptazymes
By fusing aptamer domains with catalytic cores, researchers have developed allosterically regulated RCDs for:
- Thrombin: Using dual-aptamer-linked split DNAzymes
- Prostate-specific antigen (PSA): Aptamer-controlled activation of RCDs
- VEGF and ATP: Loop-based hairpin reconfiguration enabling signal release
These constructs typically yield detection limits in the nanomolar to sub-nanomolar range and can be configured for fluorescence or colorimetric output.
Viral RNA and miRNA Detection: Precision Tools for Pandemic Response
SARS-CoV-2 Detection
- 10–23 and X10–23 DNAzymes have been central in COVID-19 diagnostics. Key developments include:
- REVEALR Platform: Achieved 20 aM sensitivity in patient NPS samples.
- Variant Discrimination: Competitive cleavage assays distinguish SARS-CoV-2 VOCs via dual fluorophore readouts.
- Saliva-Based Assays: Quasi-exponential RCA initiated by 10–23 cleavage allows viral RNA detection in minimally processed saliva with 86% sensitivity and 100% specificity.
miRNA and Genomic RNA Targets
The structure-specific recognition capability of RCDs also enables direct cleavage of highly structured RNA, such as microRNAs and viral genomes, providing a route for decentralized nucleic acid testing without reverse transcription.
Clinical Performance and Translation Challenges
Clinical Data from Human Samples
RCD-based assays have been validated across diverse clinical matrices:
| Urine |
RFD-EC1 and aRCD-EC1 achieved 100% sensitivity in UTI detection. |
| Sputum |
EPDz20 M5 distinguished eosinophilic from non-eosinophilic asthma with 96–100% accuracy. |
| Tumor Tissue |
AA12-5 differentiated malignant from benign breast tumors with 92% sensitivity. |
Limitations and Stability Concerns
Challenges hindering commercialization include:
- Target ambiguity in cell-based selection protocols.
- Sensitivity to nucleases, particularly in complex biological samples.
- Sample preparation requirements often involve centrifugation, lysis, or heat treatment.
Strategies to address these issues involve buffer engineering (low pH, monovalent ions), use of nuclease-resistant analogs (e.g., FANA), and integration with automated processing systems.
Future Outlook: From Lab Innovation to Clinical Standard
To unlock the full potential of RCDs in diagnostics, several developments are underway:
- Miniaturized and integrated platforms: Electrochemical and lateral flow formats reduce equipment needs and enable field-deployable testing.
- Rapid, automated selection pipelines: Robotics and machine learning may reduce SELEX rounds from 20+ to under 10.
- Expansion of target libraries: Direct selection against validated biomarkers will enhance regulatory compliance and clinical adoption.
- Commercial pathways: Early efforts from Alpha Measure and GlucoSentient show promise but require scale-up and ISO-certified production.
Conclusion
RNA-cleaving DNAzymes have emerged as agile, precise, and powerful tools for next-generation clinical diagnostics. Their adaptability across platforms, compatibility with multiple biomarkers, and resilience in diverse biological matrices provide a unique foundation for the future of POC diagnostics. As technical challenges are addressed through innovation and integration, RCD-based assays are poised to become a cornerstone in molecular diagnostics, delivering rapid, reliable, and accessible solutions for global health.
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Reference
- Ali, Monsur, et al. "In-vitro Clinical Diagnostics using RNA-Cleaving DNAzymes." ChemBioChem 25.11 (2024): e202400085.
This article is for research use only. Do not use in any diagnostic or therapeutic application.
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