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Target-site selection for the 10-23 DNAzyme
Murray J Cairns1, Lun-Quan Sun
1Johnson & Johnson Research Laboratories, Australian Technology Park, Eveleigh.
Methods in Molecular Biology (Clifton, N.J.)
|March 16, 2004
Summary
A new multiplex assay efficiently screens many DNAzymes for RNA cleavage. This method identifies accessible RNA sites for gene suppression strategies, finding 10% of tested DNAzymes highly effective.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The 10-23 DNAzyme cleaves RNA at purine-pyrimidine junctions.
- mRNA secondary structures can shield these cleavage sites from DNAzymes.
- Efficient methods are needed to select optimal DNAzymes for specific RNA targets.
Purpose of the Study:
- To develop a multiplex assay for high-throughput screening of DNAzyme activity.
- To identify effective DNAzyme cleavage sites within the HPV16 E6/E7 transcript.
- To assess the utility of this assay for discovering accessible RNA regions for gene suppression.
Main Methods:
- Development of a multiplex assay for simultaneous analysis of over 50 DNAzymes.
- Evaluation of 80 DNAzyme sites on a full-length HPV16 E6/E7 transcript.
- Comparative analysis of selected DNAzymes in conventional assays.
Main Results:
- The multiplex assay enabled efficient screening of numerous DNAzymes.
- Approximately 10% of tested DNAzymes demonstrated substantial cleavage of the long RNA transcript.
- Identified DNAzyme-sensitive regions are potentially accessible to other RNA-targeting agents.
Conclusions:
- The multiplex assay is effective for optimizing DNAzyme selection and target-site identification.
- This method can locate accessible sites on long RNA transcripts for gene suppression.
- The approach is valuable for developing nucleic acid-based therapeutic strategies.