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Kanezaki, Hiroshi,Nishimoto, Sei-Ichi,Tanabe, Kazuhito Korean Society of Photoscience 2014 Rapid communication in photoscience Vol.3 No.4
We characterized the one-electron reduction of oligodeoxynucleotides with a 2-oxopropyl group on a thymine base ($d^{oxo}T$) and applied the reaction to the radiolytic activation of DNAzyme function. We designed a system in which the DNAzyme function of cleaving mRNA was suppressed by introduction of $d^{oxo}T$ into the strand of DNAzyme. Hypoxic X-irradiation led to recovery of the cleavage ability because the 2-oxopropyl group was removed to form unmodified DNAzyme. We characterized the DNAzyme function by monitoring the fluorescence change of fluorophore- and quencher-labeled target strands. We confirmed that the DNAzyme function could be regulated by hypoxic X-irradiation and the reaction of $d^{oxo}T$.
Kim, J.E.,Yoon, S.,Mok, H.,Jung, W.,Kim, D.E. North-Holland Pub ; Elsevier Science Ltd 2012 FEBS letters Vol.586 No.21
RNA-cleaving DNAzymes were constructed to target the point mutation in the BCR-ABL transcript that causes imatinib resistance in leukemic cells. We examined the effect of 12mer peptide nucleic acids (PNAs) as facilitator oligonucleotides that bind to RNA substrate at the termini of the DNAzyme to improve DNAzyme-mediated cleavage of full-length RNA. When imatinib-resistant cells were transfected with the facilitator PNA and DNAzyme, DNAzyme activity was enhanced and the cells were sensitized to imatinib treatment. Thus, facilitator PNA may be used to enhance activity of antisense oligonucleotide targeting the full-length transcript.
A Reusable Pb<sup>2+</sup> Detecting Aptasensor Employing a Gold Nanorod-DNAzyme Conjugate
Lee, Jayeon,Ha, Tai Hwan The Korean Vacuum Society 2015 Applied Science and Convergence Technology Vol.24 No.5
Here, we demonstrated a $Pb^{2+}$ detecting aptasensor using $Pb^{2+}$-sensitive DNAzyme-conjugated gold nanorods (GNRs). Fluorescent DNA substrates that were initially quenched by GNRs, are released in response to $Pb^{2+}$ ions to give a substantial fluorescence signal. The GNR-tethered DNAzyme is reusable at least three times with a LOD of 50 nM.
Washing-free Electrochemical Strategy to Detect Target DNA Utilizing Peroxidase Mimicking DNAzyme
이상모,Sujeong Shin,김효용,원병연,안준기,박기수,박현규 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.5
We herein describe a novel washing-free electrochemical strategy for target DNA detection by utilizing the peroxidase mimicking DNAzyme. The DNAzymeincorporated DNA probes, the key components of this strategy, are designed to be not able to form G-quadruplex structure in the initial state such that the peroxidase mimicking activity of DNAzyme is kept suppressed. The DNAzyme sequences, however, would be converted to the catalytically active G-quadruplex structure by the presence of target DNA, whose peroxidase mimicking activity then promote the precipitation reaction of 4-chloronaphthol (4- CN) on the electrode. Due to the precipitates produced on the electrode surface, the electrochemical reaction between the redox materials and electrode surface is inhibited, consequently leading to a significant increase of the impedance signal. Based on this novel electrochemical design principle, we successfully detected the target DNA from sexually transmitted disease (STD) pathogens such as Chlamydia trachomatis, Trichomonas vaginalis, and Herpes simplex virus type 2 in real patient samples, verifying its practical diagnostic capability in the clinical applications.
Efficient Target Site Selection for an RNA-cleaving DNAzyme through Combinatorial Library Screening
Ki-Sun Kim,Woo-Hyung Choi,Soo-Jeong Gong,오상택,Jae Hyun Kim,김동은 대한화학회 2006 Bulletin of the Korean Chemical Society Vol.27 No.5
Identification of accessible sites in targeted RNAs is a major limitation to the effectiveness of antisense oligonucleotides. A class of antisense oligodeoxynucleotides, known as the "10-23"DNA enzyme or DNAzyme, which is a small catalytic DNA, has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. We have designed a strategy to identify accessible cleavage sites in the target RNA, which is hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. A pool of DNAzymes of 58 nucleotides-length that possess randomized annealing arms, catalytic core sequence, and fixed 5'/3'-end flanking sequences was designed and screened for their ability to cleave the target RNA. The screening procedure, which includes binding of DNAzyme pool to the target RNA under inactive condition, selection and amplification of active DNAzymes, incubation of the selected DNAzymes with the target RNA, and target site identification on sequencing gels, identified 16 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in terms of kinetics and accessibility. These selected DNAzymes were effective in cleaving the target RNA in the presence of Mg2+. This strategy can be applicable to identify accessible sites in any target RNA for antisense oligonucleotides-based gene inactivation methods.
Efficient Target Site Selection for an RNA-cleaving DNAzyme through Combinatorial Library Screening
Kim, Ki-Sun,Choi, Woo-Hyung,Gong, Soo-Jeong,Oh, Sang-taek,Kim, Jae-Hyun,Kim, Dong-Eun Korean Chemical Society 2006 Bulletin of the Korean Chemical Society Vol.27 No.5
Identification of accessible sites in targeted RNAs is a major limitation to the effectiveness of antisense oligonucleotides. A class of antisense oligodeoxynucleotides, known as the “10-23” DNA enzyme or DNAzyme, which is a small catalytic DNA, has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. We have designed a strategy to identify accessible cleavage sites in the target RNA, which is hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. A pool of DNAzymes of 58 nucleotides-length that possess randomized annealing arms, catalytic core sequence, and fixed 5'/3'-end flanking sequences was designed and screened for their ability to cleave the target RNA. The screening procedure, which includes binding of DNAzyme pool to the target RNA under inactive condition, selection and amplification of active DNAzymes, incubation of the selected DNAzymes with the target RNA, and target site identification on sequencing gels, identified 16 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in terms of kinetics and accessibility. These selected DNAzymes were effective in cleaving the target RNA in the presence of $Mg^{2+}$. This strategy can be applicable to identify accessible sites in any target RNA for antisense oligonucleotides-based gene inactivation methods.
Chemiluminescence competitive aptamer assay for the detection of aflatoxin B1 in corn samples
Shim, W.B.,Mun, H.,Joung, H.A.,Ofori, J.A.,Chung, D.H.,Kim, M.G. Butterworths ; Taylor Francis ; Elsevier Science 2014 FOOD CONTROL Vol.36 No.1
In this study, we developed a chemiluminescence competitive aptamer assay for aflatoxin B1 (AFB1) using a hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) linked with an aptamer specific to AFB1. Single, double, and triple HRP-DNAzymes coupled to the AFB1 aptamer were tested, and the AFB1 aptamer linked with double HRP-DNAzymes that produced sufficient chemiluminescence (CL) values when binding to AFB1-ovalbumin (OVA) used as a coating antigen, was selected. Under conditions optimized by testing key parameters, the aptamer assay exhibited a wide dynamic range from 0.1 to 10 ng/mL and showed a limit of detection of 0.11 ng/mL. Cross-reaction to aflatoxin G1, aflatoxin M1, and zearalenone was observed but no cross-reaction to other mycotoxins or the herbicide (atrazine) was observed. Aqueous methanol (20%) gave a good extraction efficiency and the matrix influence from corn extracts was successfully reduced through 4-fold dilution with water. Recovery from spiked corn samples averaged from 60.4 to 105.5%. Thus, the aptamer linked with HRP-DNAzymes can be useful as a reagent in the development of a biosensor for the rapid and simple detection of AFB1. Results from this study provide the basis for research into the development of various aptasensors for AFB1 analysis in foods.
A Reusable Pb<SUP>2+</SUP> Detecting Aptasensor Employing a Gold Nanorod-DNAzyme Conjugate
Jayeon Lee,Tai Hwan Ha 한국진공학회(ASCT) 2015 Applied Science and Convergence Technology Vol.24 No.5
Here, we demonstrated a Pb<SUP>2+</SUP> detecting aptasensor using Pb<SUP>2+</SUP>-sensitive DNAzymeconjugated gold nanorods (GNRs). Fluorescent DNA substrates that were initially quenched by GNRs, are released in response to Pb<SUP>2+</SUP> ions to give a substantial fluorescence signal. The GNR-tethered DNAzyme is reusable at least three times with a LOD of 50 nM.