Target site selection for an RNA-cleaving catalytic DNA
| Autor: | Murray J. Cairns, Craig Witherington, Lun-Quan Sun, Toni M. Hopkins, Li Wang |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Molecular Sequence Data
Biomedical Engineering Deoxyribozyme Oligonucleotides DNA Single-Stranded Bioengineering Biology Cleavage (embryo) Applied Microbiology and Biotechnology Nucleic acid secondary structure Proto-Oncogene Proteins c-myc chemistry.chemical_compound Animals Humans Multiplex Gene Papillomaviridae Cells Cultured chemistry.chemical_classification Base Sequence RNA Muscle Smooth DNA Catalytic Oncogene Proteins Viral Rats Enzyme chemistry Biochemistry Molecular Medicine Nucleic Acid Conformation DNA Biotechnology |
| Zdroj: | Nature biotechnology. 17(5) |
| ISSN: | 1087-0156 |
| Popis: | A small catalytic DNA, known as the 10–23 DNA enzyme or deoxyribozyme, has been shown to efficiently hydrolyze RNA at purine-pyrimidine (R-Y) junctions in vitro. Although these potentially cleavable junctions are ubiquitous, they are often protected from deoxyribozyme activity by RNA secondary structure. We have developed a multiplex cleavage assay for screening the entire length of a target RNA molecule for deoxyribozyme cleavage sites that are efficient, both in terms of kinetics and accessibility. This strategy allowed us to simultaneously compare the RNA cleaving activity of 80 deoxyribozymes for a model target gene (HPV16 E6), and an additional 60 deoxyribozymes against the rat c-myc target. The human papilloma virus (HPV) target was used primarily to characterize the multiplex system and determine its validity. The c-myc target, coupled with a smooth muscle cell proliferation assay, allowed us to assess the relationship between in vitro cleavage efficiency and c-myc gene suppression in cell culture. The multiplex reaction approach streamlines the process of revealing effective deoxyribozymes in a functional assay and provides accessibility data that may also be applicable to site selection for other hybridization-based agents. |
| Databáze: | OpenAIRE |
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