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Nucleic acid mutation analysis using catalytic DNA.

Johnson and Johnson Research Laboratories, Australian Technology Park, Level 4, 1 Central Avenue, Eveleigh, NSW 1430, Australia.
Nucleic Acids Research (Impact Factor: 9.11). 03/2000; 28(3):E9.
Source: PubMed

ABSTRACT The sequence specificity of the '10-23' RNA-cleaving DNA enzyme (deoxyribozyme) was utilised to discriminate between subtle differences in nucleic acid sequence in a relatively conserved segment of the L1 gene from a number of different human papilloma virus (HPV) genotypes. DNA enzymes specific for the different HPV types were found to cleave their respective target oligoribonucleotide substrates with high efficiency compared with their unmatched counterparts, which were usually not cleaved or cleaved with very low efficiency. This specificity was achieved despite the existence of only very small differences in the sequence of one binding arm. As an example of how this methodology may be applied to mutation analysis of tissue samples, type-specific deoxyribozyme cleavable substrates were generated by genomic PCR using a chimeric primer containing three bases of RNA. The RNA component enabled each amplicon to be cleavable in the presence of its matching deoxyribozyme. In this format, the specificity of deoxyribozyme cleavage is defined by Watson-Crick interactions between one substrate-binding domain (arm I) and the polymorphic sequence which is amplified during PCR. Deoxy-ribozyme-mediated cleavage of amplicons generated by this method was used to examine the HPV status of genomic DNA derived from Caski cells, which are known to be positive for HPV16. This method is applicable to many types of nucleic acid sequence variation, including single nucleotide polymorphisms.

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    ABSTRACT: A small oligodeoxyribonucleotide derived from in vitro selection has been shown to be capable of efficient sequence-specific cleavage of RNA at purine-pyrimidine junctions. As the reaction readily takes place under simulated physiologic conditions, this molecule described as the 10-23 general purpose RNA-cleaving DNA enzyme, has potential as a therapeutic agent. To further explore the character of this prototype, we examined the influence of base substitution and binding arm length asymmetry on its RNA cleaving activity. Surprisingly, substitution of the proximal nucleotide on the 3'-arm, to allow nonstandard Watson-Crick interactions, was found in some instances to improve the cleavage reaction rate. Although the identity of the unpaired purine in the RNA substrate cleavage site was found to have only a subtle influence on the rate of catalysis, with a slight decrease observed when a G at this position was changed to an A, nucleotide substitution (G to C) in the core motif at position 14 was found to completely abolish catalysis. The effect of arm length reduction varied with RNA substrate sequence and extent of helix asymmetry. Where the cleavage rate of one substrate was impaired by truncation of the deoxyribozymes 5'-arm (6 bp), the same modification in reactions with a different sequence produced a rate enhancement. Truncation of the 3'-arm, however, had no effect on the reaction rate of the one substrate tested yet nearly halved the cleavage rate in another substrate.
    Antisense and Nucleic Acid Drug Development 11/2000; 10(5):323-32. DOI:10.1089/oli.1.2000.10.323
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    Journal of Clinical Investigation 12/2000; 106(10):1189-95. DOI:10.1172/JCI11620 · 13.77 Impact Factor