Related Experiment Videos
Target site selection for an RNA-cleaving catalytic DNA
M J Cairns1, T M Hopkins, C Witherington
1Johnson and Johnson Research Laboratories, Sydney, Australia.
Nature Biotechnology
|May 20, 1999
Summary
Deoxyribozymes efficiently cleave RNA at specific sites. A new multiplex assay identifies effective deoxyribozymes and their accessible cleavage sites on target RNA molecules.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Therapeutics
Background:
- Catalytic DNA, or deoxyribozymes (10-23 DNA enzyme), hydrolyze RNA at purine-pyrimidine junctions.
- RNA secondary structures can impede deoxyribozyme accessibility and activity.
- Efficient identification of functional deoxyribozyme sites is crucial for therapeutic applications.
Purpose of the Study:
- To develop and validate a multiplex cleavage assay for screening deoxyribozymes against target RNA.
- To assess the kinetics and accessibility of deoxyribozyme cleavage sites.
- To correlate in vitro cleavage efficiency with gene suppression in a cellular context.
Main Methods:
- Developed a multiplex assay to screen multiple deoxyribozymes against target RNA simultaneously.
- Tested 80 deoxyribozymes against HPV16 E6 RNA and 60 against rat c-myc RNA.
- Utilized a smooth muscle cell proliferation assay to evaluate c-myc gene suppression.
Main Results:
- The multiplex assay effectively screened numerous deoxyribozymes for RNA cleavage activity.
- Identified specific deoxyribozymes with high cleavage efficiency and accessibility.
- Demonstrated a correlation between in vitro cleavage and in vivo gene suppression for c-myc.
Conclusions:
- The multiplex cleavage assay is a streamlined method for discovering effective deoxyribozymes.
- This approach provides valuable data on cleavage site accessibility for hybridization-based agents.
- The findings advance the development of RNA-targeting deoxyribozymes for gene regulation.