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


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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    • "Another approach involves the identification of accessible sites with antisense oligonucleotides (e.g., Kurreck et al, 2002) or oligonucleotide libraries (Sohail et al, 2001) in combination with RNase H. Nevertheless, such approaches are not efficient and do not always predict active deoxyribozymes. (Cairns et al, 2000; Kurreck et al, 2002) For example, RNase H can act on short heteroduplexed regions (Wu et al, 1999) and, therefore, only partial heteroduplex formation between an antisense oligonucleotide and RNA can lead to cleavage of RNA. Such short sequences are unlikely to imitate the full length of the deoxyribozyme binding arms. "
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    ABSTRACT: We employed an approach using oligonucleotide scanning arrays and computational analysis to conduct a systematic analysis of the interaction between catalytic nucleic acids (DNA enzymes or DNAzymes) and long RNA targets. A radio-labelled transcript representing mRNA of Xenopus cyclin B5 was hybridised to an array of oligonucleotides scanning the first 120 nucleotides of the coding region to assess the ability of the immobilised oligonucleotides to form heteroduplexes with the target. The hybridisation revealed oligonucleotides showing varying levels of signal intensities along the length of the array, reflecting on the variable accessibility of the corresponding complementary regions in the target RNA. Deoxyribozymes targeting a number of these regions were selected and tested for their ability to cleave the target RNA. The mRNA cleavage observed indicates that indeed target accessibility was an important component in the activity of deoxyribozymes and that it was important that at least one of the two binding arms was complementary to an accessible site. Computational analysis suggested that intra-molecular folding of deoxyribozymes into stable structures may also negatively contribute to their activity. 10-23 type deoxyribozymes generally appeared more active than 8-17 type and it was possible to predict deoxyribozymes with high cleavage efficiency using scanning array hybridization and computational analysis as guides. The data presented here therefore have implications on designing effective DNA enzymes.
    Preview · Article · Jul 2006 · Journal of RNAi and Gene Silencing
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    • "The reduction in DNAzyme activity due to the mismatches in the arms varied according to the length and the mismatch position. Our results, which were obtained using long cRNA transcripts, correlated well with previous studies on the influence of arm length asymmetry and base substitutions performed on a short target substrate (Cairns et al, 2000a; Cairns et al, 2000b). We found that, in general, mismatches in arm I are more effective in reducing the enzyme activity and thus increasing the degree of selectivity, although this is not always the case. "
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    ABSTRACT: Many dominantly inherited disorders are caused by missense amino acid substitutions resulting from a single nucleotide exchange in the encoding gene. For these disorders, where proteins expressed from the mutant alleles are often pathogenic and present throughout life, gene silencing, through intervention at the mRNA level, holds promise as a therapeutic approach. We have used mutations that underlie the slow channel congenital myasthenic syndrome (SCCMS) as a model system to study allele-specific gene silencing of RNA transcripts by DNAzymes. We tested the ability of DNAzymes to give allele-specific cleavage for i) mutations that create cleavage sites, and ii) mutations located close to a DNAzyme cleavage site that create a potential mismatch in the binding arms. For both we demonstrate selective cleavage of mutant transcripts under simulated physiological conditions. For DNAzymes with binding arm mismatches the degree of selectivity for mutant over wild type may be enhanced by optimising the mismatch position as well as the binding arm length. The optimal sites for mismatches are 1.1 and 1.2 in arm I, and 16.2 in arm II. Asymmetric binding arm DNAzymes with a shorter arm I are more discriminative. Our results show it should be possible to apply DNAzyme-mediated cleavage of mutant alleles even when the mutant does not itself create a putative cleavage site. This therapeutic approach may be well suited to dominantly inherited disorders such as SCCMS, where loss of some wild type transcripts is unlikely to have pathogenic consequences.
    Full-text · Article · Jul 2005 · Journal of RNAi and Gene Silencing
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    • "RNA-cleaving deoxyribozymes have been applied for purposes beyond those mentioned above. For example, such deoxyribozymes have been used for detecting in vitro-generated RNA modifications (140); for mapping sites of in vitro RNA crosslinking (141); for mapping RNA branch points (72,142,143); for probing higher-order RNA structure (144); for quantifying nucleic acid production during PCR amplification (145); for analyzing nucleic acid sequence mutations (146); for detecting specific microbial rRNAs (147); for constructing logical computation circuits (148–151); and for manipulating a DNA-based nanodevice (152). A deoxyribozyme has been identified that cleaves unnatural RNA linkages, which may be useful for certain biotechnology applications (153). "
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    ABSTRACT: Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from random-sequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg2+ to catalyze attack by a specific RNA 2′-hydroxyl group on the adjacent phosphodiester linkage, forming a 2′,3′-cyclic phosphate and a 5′-hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.
    Preview · Article · Feb 2005 · Nucleic Acids Research
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