Mechanism and Utility of an RNA-Cleaving DNA Enzyme

Santoro, Stephen W.; Joyce, Gerald F.

doi:10.1021/bi9812221

Cited by 419 publications

(533 citation statements)

References 47 publications

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“…Two kinds of catalytic DNA sequences, RNA-cleaving DNAzyme (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) 26,27 and G-quadruplex horseradish peroxidase-mimicking DNAzyme (PW17) [28][29][30] were applied in our research. The 10-23 RNAcleaving DNAzyme has been reported to cleave any purinepyrimidine (RY) junction under simulated physiological conditions 26 ; hence, it was applied in our strategy for the cleaving of target RNA molecule to initiate the following exponential amplification.…”

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confidence: 99%

Cleavage-based signal amplification of RNA

2013

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RNA detection has become an integral part of current biomedical research. Up to now, the reverse transcription-PCR has been the most practical method to detect mRNA targets. However, RNA detection by reverse transcription-PCR requires sophisticated equipment and it is highly sensitive to contamination with genomic DNA. Here we report a new isothermal reaction to simultaneously amplify and detect RNA, based on cleavage by DNAzyme and signal amplification. Cleavage-based signal amplification of RNA cannot be contaminated by genomic DNA and is suitable for the detection of both mRNA and microRNA targets, with high specificity and sensitivity. Moreover, the detection results can be reported in a colorimetric or real-time fluorometric way for different detection purposes.

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confidence: 99%

Cleavage-based signal amplification of RNA

2013

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“…As shown in Figure 3, DZ7 effectively cleaved b-gal RNA to produce the products with the expected sizes (650 nt and 300 nt) ( Figure 3). It has been reported that the 10-23 DNA enzyme is almost completely intolerant of sequence changes within the catalytic domain 7 and a single substitution from T to G in the catalytic domain of DNA enzyme abolishes target RNA cleavage activity. 17 As shown in Figure 3, DZ7m with the same substitution had no cleavage activity.…”

Section: Construction Of the Pssxe Ssdna Expression Vectormentioning

confidence: 99%

“…One such catalytic ODN referred as 10-23 DNA enzyme has the potential to cleave almost any RNA target containing purine-pyrimidine junctions. 6,7 This 10-23 DNA enzyme, isolated by in vitro selection using a combinatorial ODN library, consists of a 15 deoxynucleotide catalytic domain flanked by two target RNA-binding domains of seven or eight deoxynucleotides each. 6,7 Unlike antisense ODNs that mainly rely on cellular Ribonuclease H activity to destroy target mRNA, the 10-23 DNA enzyme is responsible for both target recognition and cleavage of target mRNA.…”

Section: Introductionmentioning

confidence: 99%

“…6,7 This 10-23 DNA enzyme, isolated by in vitro selection using a combinatorial ODN library, consists of a 15 deoxynucleotide catalytic domain flanked by two target RNA-binding domains of seven or eight deoxynucleotides each. 6,7 Unlike antisense ODNs that mainly rely on cellular Ribonuclease H activity to destroy target mRNA, the 10-23 DNA enzyme is responsible for both target recognition and cleavage of target mRNA. 6,7 This 10-23 DNA enzyme has been evaluated in a number of studies both in vitro and in vivo for the suppression of various target gene expression and has shown promising results.…”

Section: Introductionmentioning

confidence: 99%

“…6,7 Unlike antisense ODNs that mainly rely on cellular Ribonuclease H activity to destroy target mRNA, the 10-23 DNA enzyme is responsible for both target recognition and cleavage of target mRNA. 6,7 This 10-23 DNA enzyme has been evaluated in a number of studies both in vitro and in vivo for the suppression of various target gene expression and has shown promising results. 8 Instead of delivering oligonucleotides externally into cells, we have developed a vector-mediated approach to produce ODNs or single-stranded DNA (ssDNA) molecules intracellularly.…”

Section: Introductionmentioning

confidence: 99%

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Intracellular production of DNA enzyme by a novel single-stranded DNA expression vector

Chen

McMicken

2003

Gene Ther

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A set of single-stranded DNA (ssDNA) expression vectors, which can generate intracellularly any ssDNA or oligodeoxynucleotide (ODN) molecules, have been developed in our laboratory. Studies from our laboratory as well as our collaborators demonstrated that these ssDNA expression vectors are capable of producing: (1) 10-23 DNA enzyme for downregulating c-raf kinase gene expression and (2) triplexforming oligodeoxynucleotide (TFO) for inducing genomic recombination. We report here the construction of a new version of ssDNA expression vector. A b-galactosidase (bgal) reporter gene was used as a test target so that the alteration of gene expression can be easily measured using b-gal activity assay. We designed a 10-23 DNA enzyme molecule that specifically cleaves b-gal mRNA at protein translation starting site (ATG). Using a cell-free RNA cleavage assay, we confirmed that this DNA enzyme molecule could effectively cleave b-gal RNA. However, a single substitution from T to G in the catalytic domain of this DNA enzyme molecule abolished its RNA cleavage activity. We also constructed an expression vector that can generate DNA enzyme molecules in cells. A549 lung carcinoma cells were cotransfected with both DNA enzyme expression vector and the b-gal reporter gene. Compared to the cells that were transfected with the mutated DNA enzyme expression vector, significant reduction of b-gal gene expression (up to 76%) was observed in the cells transfected with DNA enzyme expression vector as indicated by the protein expression level as well as its enzyme activity. These results further suggest that the ssDNA expression vector has potential applications in the study of gene function and target validation.

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Antiviral Agents, RNA Viruses (Other than HIV), and Orthopoxviruses

Tseng

Laughlin

2003

Burger's Medicinal Chemistry and Drug Discovery

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Since our last review on antiviral agents for RNA viruses other than HIV, significant advances have occurred in the field of antiviral chemotherapy. The U.S. Food and Drug Administration (FDA) approved two new drugs (Relenza and Tamiflu) for the treatment of influenza for both adults and children. Pegylated interferon has improved the efficacy of interferon, particularly in combination with ribavirin for the treatment of chronic hepatitis C infections. A monoclonal antibody, Synagis, has been approved for the prevention of serious lower respiratory tract infections in high‐risk pediatric patients. A new drug application (NDA) has also been submitted to the FDA for the treatment of infections caused by rhinoviruses and enteroviruses. This chapter will update the discovery, preclinical evaluation, and clinical data supporting the development of antiviral agents for RNA viruses other than HIV. In addition, as a component of biodefense‐related activities, this chapter will review the historical and current development of antiviral agents for orthopoxviruses.

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Mechanism and Utility of an RNA-Cleaving DNA Enzyme

Cited by 419 publications

References 47 publications

Cleavage-based signal amplification of RNA

Cleavage-based signal amplification of RNA

Intracellular production of DNA enzyme by a novel single-stranded DNA expression vector

Antiviral Agents, RNA Viruses (Other than HIV), and Orthopoxviruses

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