Multiple‐Catalytic Sensing of Nucleic Acid Sequences by Utilising a DNA–RNA–DNA Chimeric Antisense Probe and RNase H with a Eukaryotic Cell‐Free Translation System

Ogawa, Atsushi

doi:10.1002/cbic.201000744

Cited by 12 publications

(5 citation statements)

References 32 publications

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“…Therefore, the use of RNase H for amplified DNA detection requires designing of RNA-based or RNA-containing probes. Both RNA and chimeric DNA–RNA–DNA molecular beacons have been used as probes. , The hybridization of a target DNA with a loop of molecular beacons formed a DNA–RNA heteroduplex, leading to the cleavage of the loop of molecular beacons and recycling of the target DNA. Therefore, the target DNA resulted in repeated cleavage of many molecular beacons.…”

Section: Homogeneous Binding Assays Incorporating Isothermal Amplific...mentioning

confidence: 99%

DNA-Mediated Homogeneous Binding Assays for Nucleic Acids and Proteins

et al. 2012

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Section: Homogeneous Binding Assays Incorporating Isothermal Amplific...mentioning

confidence: 99%

DNA-Mediated Homogeneous Binding Assays for Nucleic Acids and Proteins

et al. 2012

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“…DNA detection, 17,18 removal of mRNA from an mRNA:cDNA duplex, RNA isotope labeling and specic cleavage of RNA in synthetic DNA. 19,20 Until now, the active site and key structural elements necessary for RNA hydrolysis have been recognized through structural analysis of RNase H, [21][22][23][24][25][26][27] while biochemical evidence for the function of JRNase is still much limited. In order to explore the mechanism of junction cleavage reaction, developing simple and efficient methods for JRNase activity assay of RNase H still remains to be done.…”

Section: Introductionmentioning

confidence: 99%

Real time monitoring of junction ribonuclease activity of RNase H using chimeric molecular beacons

Liu

Xiang

Long

et al. 2013

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As a highly conserved damage repair protein, except for hydrolysis of DNA-RNA heteroduplex endonucleolytically, RNase H can cleave RNA-DNA junctions in Okazaki fragment processing through its junction ribonuclease (JRNase) activity. We report here a real time fluorescence method for detecting JRNase activity of RNase H with high accuracy by applying chimeric molecular beacons as substrates. The detection limit of E. coli RNase H is 0.5 U ml(-1). The Km and kcat are 20 nM and 0.6 s(-1), respectively. In addition, we used the method to investigate the effect of chemical drugs on the enzyme and found that both penicillin and streptomycin sulfate inhibit its activity with the IC50 values of 0.2 and 0.07 mM, respectively. Finally, we applied the method to reliably detect the JRNase level in tumor cells. In summary, these data indicate that the simple, rapid and sensitive method can be hopefully applied for high-throughput detection of samples and drug screening in vitro.

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“…Contrary to the traditional hybridization assay with the target-to-signal ratio of 1:1, target recycling-oriented amplification makes one target function as a “catalyst” to interact with multiple probes, achieving great signal amplification . So far, a variety of nucleases such as Rnase H, nicking endonuclease, , flap endonuclease, DNase I, duplex-specific nuclease, endonuclease IV, exonuclease III, and lambda exonuclease have been employed as the cleavage enzymes in the target recycling-oriented amplification for sensitive detection of DNA, RNA, and proteins. Among them, exonuclease III (Exo III) is frequently used because of its easy availability, high catalytic activity, and wide applicability .…”

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confidence: 99%

Rapid and Label-Free Monitoring of Exonuclease III-Assisted Target Recycling Amplification

et al. 2012

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Target recycling-oriented amplification has been widely applied for sensitive detection of DNA, RNA, and proteins due to its successful overcoming the inherent limitation of target-to-signal ratio of 1:1 in the traditional hybridization assay. Exonuclease III (Exo III) is usually used as the cleavage enzyme in the target recycling-oriented amplification because of its easy availability, high catalytic activity, and wide applicability. Even though Exo III is assumed to be double-stranded DNA (dsDNA) specific exonuclease in most literature, its cleavage of single-strand DNA (ssDNA) does occur, resulting in the target-independent degradation of probes. Herein, we design an intramolecular displacement probe with the capability of resistance to the nonspecific digestion of Exo III and fast hybridization kinetics. Through the substitution of 2-aminopurine for adenine in the intramolecular displacement probes, we develop a rapid and label-free approach to monitor Exo III-assisted target recycling amplification. We further demonstrate that this method can be used for the detection of DNA and proteins with excellent specificity and high sensitivity. Importantly, this method can be extended to rapid, label-free and multiplexed detection of various nucleic acids, proteins, and small molecules using different kinds of fluorescent nucleotide analogues and specific aptamers.

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Multiple‐Catalytic Sensing of Nucleic Acid Sequences by Utilising a DNA–RNA–DNA Chimeric Antisense Probe and RNase H with a Eukaryotic Cell‐Free Translation System

Cited by 12 publications

References 32 publications

DNA-Mediated Homogeneous Binding Assays for Nucleic Acids and Proteins

DNA-Mediated Homogeneous Binding Assays for Nucleic Acids and Proteins

Real time monitoring of junction ribonuclease activity of RNase H using chimeric molecular beacons

Rapid and Label-Free Monitoring of Exonuclease III-Assisted Target Recycling Amplification

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