The Experts below are selected from a list of 1155 Experts worldwide ranked by ideXlab platform
Michal Hocek - One of the best experts on this subject based on the ideXlab platform.
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Polymerase Synthesis of Base-Modified DNA
Modified Nucleic Acids, 2016Co-Authors: Jitka Dadová, Hana Cahová, Michal HocekAbstract:Enzymatic synthesis of base-modified DNA by polymerase incorporation of modified nucleotides is discussed. Modified 2′-deoxyribonucleoside triphosphates (dNTPs) are key substrates for polymerases and can be prepared either by triphosphorylation of modified nucleosides or by direct aqueous cross-coupling reactions of halogenated dNTPs with alkynes, arylboronic acids, or alkenes. The methods of polymerase synthesis include primer extension, PCR, Nicking Enzyme amplifications, and other methods which enable the synthesis of diverse types of long or short and double-stranded DNA or single-stranded oligonucleotides. The applications include labeling in diagnostics (labeling or coding of DNA bases) and chemical biology (bioconjugations, modulation of protein binding, etc.).
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Polymerase synthesis of DNA labelled with benzylidene cyanoacetamide-based fluorescent molecular rotors: fluorescent light-up probes for DNA-binding proteins.
Chemical communications (Cambridge England), 2015Co-Authors: Dmytro Dziuba, Radek Pohl, Michal HocekAbstract:Viscosity-sensitive fluorophores, fluorescent molecular rotors based on aminobenzylidene–cyanoacetamide moiety, were tethered to 2′-deoxycytidine triphosphate via a propargylamine linker and incorporated into DNA by polymerases in primer extension, Nicking Enzyme amplification or PCR. DNA probes incorporating modified nucleosides show a light-up response upon binding to a protein.
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Fluorescence quenching in oligonucleotides containing 7-substituted 7-deazaguanine bases prepared by the Nicking Enzyme amplification reaction.
Bioconjugate chemistry, 2015Co-Authors: Petra Ménová, Dmytro Dziuba, Pedro Güixens-gallardo, Piotr Jurkiewicz, Martin Hof, Michal HocekAbstract:Recently, we reported the use of the Nicking Enzyme Amplification Reaction (NEAR) for the enzymatic synthesis of short oligonucleotides (ONs) containing 5-substituted pyrimidine or 7-substituted 7-deazaadenine nucleotides. Since no oligonucleotide products were visible on agarose gels stained by an intercalating dye (GelRed), we assumed that the method did not work for 7-substituted 7-deazaguanine deoxyribonucleoside triphosphates. We revisited the work and found that the NEAR method works for 7-deazaguanine nucleotides as well but that the resulting modified ONs quench the fluorescence of DNA intercalators, rendering them invisible on gel electrophoresis stained by them. Here, we report on the modified methodology for the NEAR synthesis and analysis of G-modified ONs and on quantification of the fluorescence quenching.
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Fluorescence Quenching in Oligonucleotides Containing 7‑Substituted 7‑Deazaguanine Bases Prepared by the Nicking Enzyme Amplification Reaction
2015Co-Authors: Petra Ménová, Dmytro Dziuba, Piotr Jurkiewicz, Martin Hof, Pedro Güixens-gallardo, Michal HocekAbstract:Recently, we reported the use of the Nicking Enzyme Amplification Reaction (NEAR) for the enzymatic synthesis of short oligonucleotides (ONs) containing 5-substituted pyrimidine or 7-substituted 7-deazaadenine nucleotides. Since no oligonucleotide products were visible on agarose gels stained by an intercalating dye (GelRed), we assumed that the method did not work for 7-substituted 7-deazaguanine deoxyribonucleoside triphosphates. We revisited the work and found that the NEAR method works for 7-deazaguanine nucleotides as well but that the resulting modified ONs quench the fluorescence of DNA intercalators, rendering them invisible on gel electrophoresis stained by them. Here, we report on the modified methodology for the NEAR synthesis and analysis of G-modified ONs and on quantification of the fluorescence quenching
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Bodipy-Labeled Nucleoside Triphosphates for Polymerase Synthesis of Fluorescent DNA
Bioconjugate chemistry, 2014Co-Authors: Dmytro Dziuba, Radek Pohl, Michal HocekAbstract:New fluorescent nucleosides and nucleoside triphosphate (dNTPs) analogs bearing the F-Bodipy fluorophore linked through a short, flexible nonconjugate tether were synthesized. The Bodipy-labeled dNTPs were substrates for several DNA polymerases which incorporated them into DNA in primer extension, Nicking Enzyme amplification reaction, and polymerase chain reaction. The fluorescence of F-Bodipy is not quenched upon incorporation in DNA and can be detected both in solutions and on gels.
Xiaojuan Liu - One of the best experts on this subject based on the ideXlab platform.
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fluorescence biosensing strategy based on mercury ion mediated dna conformational switch and Nicking Enzyme assisted cycling amplification for highly sensitive detection of carbamate pesticide
Biosensors and Bioelectronics, 2016Co-Authors: Xiuzhong Wang, Ting Hou, Shanshan Dong, Xiaojuan LiuAbstract:Pesticides are of great importance in agricultural and biological fields, but pesticide residues may harm the environment and human health. A highly sensitive fluorescent biosensor for the detection of carbamate pesticide has been developed based on acetylcholinesterase (AChE)-catalyzed hydrolysis product triggered Hg(2+) release coupled with subsequent Nicking Enzyme-induced cleavage of a duplex DNA for cycling amplification. In this protocol, two DNA probes, an unmodified single-stranded helper DNA probe 1 (HP1) and a quencher-fluorophore probe (QFP) are ingeniously designed. HP1 can be folded into hairpin configuration through T-Hg(2+)-T base pair formation. QFP, labeled with FAM and BHQ1 at its two terminals, contains the recognition sequence and the cleavage site of the Nicking Enzyme. In the presence of carbamate pesticide, the activity of AChE is inhibited, and the amount of the product containing the thiol group generated by the hydrolysis reaction of acetylthiocholine chloride (ACh) decreases, resulting in the release of a low concentration of Hg(2+). The number of HP1 that can be selectively unfolded would be reduced and the subsequent Nicking Enzyme-assisted cleavage processes would be affected, resulting in decreased fluorescence signals. The fluorescence intensity further decreases with the increase of the pesticide concentration. Therefore, the pesticide content can be easily obtained by monitoring the fluorescence signal change, which is inversely proportional to the logarithm of the pesticide concentration. The detection limit of aldicarb, the model analyte, is 3.3 μgL(-1), which is much lower than the Chinese National Standards or those previously reported. The as-proposed method has also been applied to detect carbamate pesticide residues in fresh ginger and artificial lake water samples with satisfactory results, which demonstrates that the method has great potential for practical application in biological or food safety field.
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Sensitive detection of T4 polynucleotide kinase activity based on coupled exonuclease reaction and Nicking Enzyme-assisted fluorescence signal amplification.
Analytical and bioanalytical chemistry, 2014Co-Authors: Ting Hou, Xiuzhong Wang, Xiaojuan LiuAbstract:As a prominent member of the 5′-kinase family, T4 polynucleotide kinase (PNK) plays an important role in gene function regulations, and the study of PNK activity and its potential inhibitors is significant for research related to the DNA phosphorylation process. Here, we proposed a novel strategy for the detection of PNK activity and its inhibition, which combines exonuclease Enzyme reaction and Nicking Enzyme-assisted fluorescence signal amplification. Through recycling cleavage of DNA fluorescence probe for signal amplification, a highly sensitive PNK sensing platform is developed, and a very low detection limit of 0.05 mU/mL is achieved, which is better than or comparable to that of the previously reported PNK assays. The present approach adopts a simple separation-free procedure in which the Enzyme assay is conducted in homogeneous solutions. Additionally, the inhibitory effects of several known kinase inhibitors on PNK have been successfully detected. Since the proposed assay exhibits the advantages of high sensitivity and simplicity, it holds great potential in providing a promising platform for convenient and highly sensitive detection of PNK activity and its inhibitors.
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Label-free colorimetric assay for base excision repair Enzyme activity based on Nicking Enzyme assisted signal amplification.
Biosensors & bioelectronics, 2013Co-Authors: Xiaojuan Liu, Xiuzhong Wang, Ting Hou, Mingqin Chen, Shufeng LiuAbstract:Specific and sensitive detection of base excision repair Enzyme activity is essential to many fundamental biochemical process researches. Here, we propose a novel label-free homogeneous strategy for visualized uracil DNA glycosylase (UDG) activity assay based on Nicking Enzyme assisted signal amplification. In this method two hairpin probes were employed for the colorimetric detection, namely hairpin probe 1 (HP 1) carrying two uracil residues in the stem, and hairpin probe 2 (HP 2) containing a G-riched DNAzyme segment, and the recognition sequence as well as the cleavage site for the Nicking Enzyme. In the presence of UDG, the uracil bases in the stem of HP 1 can be specifically recognized and hydrolyzed by UDG, which leads to the destabilization of its stem containing abasic sites (AP sites), and then results in the opening of HP 1 to form a single strand. The opened HP 1 hybridizes with HP 2 to form a DNA duplex, which initiates the specific cleavage of HP 2 by the Nicking Enzyme, leading to the release of G-riched DNAzyme segments. As a result, HP 1 is released and able to hybridize with another HP 2 to induce the continuous cleavage of HP 2, generating enormous amount of G-riched DNAzyme segments. Finally, the G-riched DNAzyme segments bind hemin to form a catalytically active G-quadruplex–hemin DNAzyme which can catalyze the H2O2-mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−) to the colored ABTS–, providing a visible signal for UDG activity detection. This assay exhibits several advantages such as simplicity, low-cost, high selectivity and desirable sensitivity, which shows great potential of providing a promising platform for convenient and visualized analysis of UDG or other biomolecules.
Jian-ding Qiu - One of the best experts on this subject based on the ideXlab platform.
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Target-Triggering Multiple-Cycle Amplification Strategy for Ultrasensitive Detection of Adenosine Based on Surface Plasma Resonance Techniques
2015Co-Authors: Gui-hong Yao, Ru-ping Liang, Chun-fang Huang, Li Zhang, Jian-ding QiuAbstract:An ultrasensitive protocol for surface plasma resonance (SPR) detection of adenosine is designed with the aptamer-based target-triggering cascade multiple cycle amplification, and streptavidin-coated Au-NPs (Au NPs–SA) enhancement to enhance the SPR signals. The cascade amplification process consists of the aptamer-based target-triggering Nicking Enzyme signaling amplification (T-NESA), the Nicking Enzyme signaling amplification (NESA) and the hybridization chain reaction (HCR), the entire circle amplification process is triggered by the target recognition of adenosine. Upon recognition of the aptamer to target adenosine, DNA s1 is released from the aptamer and then hybridizes with hairpin DNA (HP1). The DNA s1 can be dissociated from HP1 under the reaction of Nicking endonuclease to initiate the next hybridization and cleavage process. Moreover, the products of the upstream cycle (T-NESA) (DNA s2 and s3) could act as the “DNA trigger” of the downstream cycle (NESA and HCR) to generate further signal amplification, resulting in the immobilization of abundant Au NPs−SA on the gold substrate, and thus significant SPR enhancement is achieved due to the electronic coupling interaction between the localized surface plasma of Au NPs and the surface plasma wave. This detection method exhibits excellent specificity and sensitivity toward adenosine with a detection limit of 4 fM. The high sensitivity and specificity make this method a great potential for detecting biomolecules with trace amounts in bioanalysis and clinical biomedicine
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Target-Triggering Multiple-Cycle Amplification Strategy for Ultrasensitive Detection of Adenosine Based on Surface Plasma Resonance Techniques
Analytical chemistry, 2014Co-Authors: Gui-hong Yao, Ru-ping Liang, Chun-fang Huang, Li Zhang, Jian-ding QiuAbstract:An ultrasensitive protocol for surface plasma resonance (SPR) detection of adenosine is designed with the aptamer-based target-triggering cascade multiple cycle amplification, and streptavidin-coated Au-NPs (Au NPs–SA) enhancement to enhance the SPR signals. The cascade amplification process consists of the aptamer-based target-triggering Nicking Enzyme signaling amplification (T-NESA), the Nicking Enzyme signaling amplification (NESA) and the hybridization chain reaction (HCR), the entire circle amplification process is triggered by the target recognition of adenosine. Upon recognition of the aptamer to target adenosine, DNA s1 is released from the aptamer and then hybridizes with hairpin DNA (HP1). The DNA s1 can be dissociated from HP1 under the reaction of Nicking endonuclease to initiate the next hybridization and cleavage process. Moreover, the products of the upstream cycle (T-NESA) (DNA s2 and s3) could act as the “DNA trigger” of the downstream cycle (NESA and HCR) to generate further signal ampl...
Chunhai Fan - One of the best experts on this subject based on the ideXlab platform.
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Target-triggered three-way junction structure and polymerase/Nicking Enzyme synergetic isothermal quadratic DNA machine for highly specific, one-step, and rapid microRNA detection at attomolar level
Analytical chemistry, 2014Co-Authors: Qing Zhang, Yongxi Zhao, Feng Chen, Chunhai FanAbstract:MicroRNAs (miRNAs) play important roles in many biological processes and are regarded as promising cancer biomarkers. Herein, a highly specific, one-step, and rapid miRNAs detection strategy with attomolar sensitivity has been developed on the basis of a target-triggered three-way junction (3-WJ) structure and polymerase/Nicking Enzyme synergetic isothermal quadratic DNA machine (ESQM). To this end, 3-WJ probes (primer and template) are designed to selectively recognize target miRNA and form the stable 3-WJ structure to trigger ESQM, resulting in a high quadratic amplified signal. A high specificity is demonstrated by the excellent discrimination of even single-base mismatched homologous sequences with mismatched bases in varied locations (close to the 3'-end, the 5'-end, and the middle). In addition, a low detection limit down to 2 amol was achieved within 30 min. This sensitivity is much higher than those of most linear amplification-based approaches and is even comparable to those of some exponential amplification-based methods. Furthermore, the applicability of this method in complex samples was demonstrated by the analysis of cancer cell small RNA extracts, results of which were in good agreement with those obtained by a commercial miRNA kit and previously published data. The miRNA with a 3' end modification (2'-O-methylation), such as plant miRNA, was also successfully detected, confirming the good universality of the proposed strategy. It is worthwhile to point out that several well-established methods using miRNA as primer for polymerization reaction are of relatively poor performance in the analysis of these modified miRNA. Therefore, these merits endow the developed strategy with powerful implications for biological research and an effective diagnostic assay.
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target triggered three way junction structure and polymerase Nicking Enzyme synergetic isothermal quadratic dna machine for highly specific one step and rapid microrna detection at attomolar level
Analytical Chemistry, 2014Co-Authors: Qing Zhang, Yongxi Zhao, Feng Chen, Chunhai FanAbstract:MicroRNAs (miRNAs) play important roles in many biological processes and are regarded as promising cancer biomarkers. Herein, a highly specific, one-step, and rapid miRNAs detection strategy with attomolar sensitivity has been developed on the basis of a target-triggered three-way junction (3-WJ) structure and polymerase/Nicking Enzyme synergetic isothermal quadratic DNA machine (ESQM). To this end, 3-WJ probes (primer and template) are designed to selectively recognize target miRNA and form the stable 3-WJ structure to trigger ESQM, resulting in a high quadratic amplified signal. A high specificity is demonstrated by the excellent discrimination of even single-base mismatched homologous sequences with mismatched bases in varied locations (close to the 3'-end, the 5'-end, and the middle). In addition, a low detection limit down to 2 amol was achieved within 30 min. This sensitivity is much higher than those of most linear amplification-based approaches and is even comparable to those of some exponential amplification-based methods. Furthermore, the applicability of this method in complex samples was demonstrated by the analysis of cancer cell small RNA extracts, results of which were in good agreement with those obtained by a commercial miRNA kit and previously published data. The miRNA with a 3' end modification (2'-O-methylation), such as plant miRNA, was also successfully detected, confirming the good universality of the proposed strategy. It is worthwhile to point out that several well-established methods using miRNA as primer for polymerization reaction are of relatively poor performance in the analysis of these modified miRNA. Therefore, these merits endow the developed strategy with powerful implications for biological research and an effective diagnostic assay.
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Target-Triggered Three-Way Junction Structure and Polymerase/Nicking Enzyme Synergetic Isothermal Quadratic DNA Machine for Highly Specific, One-Step, and Rapid MicroRNA Detection at Attomolar Level
2014Co-Authors: Qing Zhang, Yongxi Zhao, Feng Chen, Chunhai FanAbstract:MicroRNAs (miRNAs) play important roles in many biological processes and are regarded as promising cancer biomarkers. Herein, a highly specific, one-step, and rapid miRNAs detection strategy with attomolar sensitivity has been developed on the basis of a target-triggered three-way junction (3-WJ) structure and polymerase/Nicking Enzyme synergetic isothermal quadratic DNA machine (ESQM). To this end, 3-WJ probes (primer and template) are designed to selectively recognize target miRNA and form the stable 3-WJ structure to trigger ESQM, resulting in a high quadratic amplified signal. A high specificity is demonstrated by the excellent discrimination of even single-base mismatched homologous sequences with mismatched bases in varied locations (close to the 3′-end, the 5′-end, and the middle). In addition, a low detection limit down to 2 amol was achieved within 30 min. This sensitivity is much higher than those of most linear amplification-based approaches and is even comparable to those of some exponential amplification-based methods. Furthermore, the applicability of this method in complex samples was demonstrated by the analysis of cancer cell small RNA extracts, results of which were in good agreement with those obtained by a commercial miRNA kit and previously published data. The miRNA with a 3′ end modification (2′-O-methylation), such as plant miRNA, was also successfully detected, confirming the good universality of the proposed strategy. It is worthwhile to point out that several well-established methods using miRNA as primer for polymerization reaction are of relatively poor performance in the analysis of these modified miRNA. Therefore, these merits endow the developed strategy with powerful implications for biological research and an effective diagnostic assay
Ting Hou - One of the best experts on this subject based on the ideXlab platform.
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fluorescence biosensing strategy based on mercury ion mediated dna conformational switch and Nicking Enzyme assisted cycling amplification for highly sensitive detection of carbamate pesticide
Biosensors and Bioelectronics, 2016Co-Authors: Xiuzhong Wang, Ting Hou, Shanshan Dong, Xiaojuan LiuAbstract:Pesticides are of great importance in agricultural and biological fields, but pesticide residues may harm the environment and human health. A highly sensitive fluorescent biosensor for the detection of carbamate pesticide has been developed based on acetylcholinesterase (AChE)-catalyzed hydrolysis product triggered Hg(2+) release coupled with subsequent Nicking Enzyme-induced cleavage of a duplex DNA for cycling amplification. In this protocol, two DNA probes, an unmodified single-stranded helper DNA probe 1 (HP1) and a quencher-fluorophore probe (QFP) are ingeniously designed. HP1 can be folded into hairpin configuration through T-Hg(2+)-T base pair formation. QFP, labeled with FAM and BHQ1 at its two terminals, contains the recognition sequence and the cleavage site of the Nicking Enzyme. In the presence of carbamate pesticide, the activity of AChE is inhibited, and the amount of the product containing the thiol group generated by the hydrolysis reaction of acetylthiocholine chloride (ACh) decreases, resulting in the release of a low concentration of Hg(2+). The number of HP1 that can be selectively unfolded would be reduced and the subsequent Nicking Enzyme-assisted cleavage processes would be affected, resulting in decreased fluorescence signals. The fluorescence intensity further decreases with the increase of the pesticide concentration. Therefore, the pesticide content can be easily obtained by monitoring the fluorescence signal change, which is inversely proportional to the logarithm of the pesticide concentration. The detection limit of aldicarb, the model analyte, is 3.3 μgL(-1), which is much lower than the Chinese National Standards or those previously reported. The as-proposed method has also been applied to detect carbamate pesticide residues in fresh ginger and artificial lake water samples with satisfactory results, which demonstrates that the method has great potential for practical application in biological or food safety field.
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Sensitive detection of T4 polynucleotide kinase activity based on coupled exonuclease reaction and Nicking Enzyme-assisted fluorescence signal amplification.
Analytical and bioanalytical chemistry, 2014Co-Authors: Ting Hou, Xiuzhong Wang, Xiaojuan LiuAbstract:As a prominent member of the 5′-kinase family, T4 polynucleotide kinase (PNK) plays an important role in gene function regulations, and the study of PNK activity and its potential inhibitors is significant for research related to the DNA phosphorylation process. Here, we proposed a novel strategy for the detection of PNK activity and its inhibition, which combines exonuclease Enzyme reaction and Nicking Enzyme-assisted fluorescence signal amplification. Through recycling cleavage of DNA fluorescence probe for signal amplification, a highly sensitive PNK sensing platform is developed, and a very low detection limit of 0.05 mU/mL is achieved, which is better than or comparable to that of the previously reported PNK assays. The present approach adopts a simple separation-free procedure in which the Enzyme assay is conducted in homogeneous solutions. Additionally, the inhibitory effects of several known kinase inhibitors on PNK have been successfully detected. Since the proposed assay exhibits the advantages of high sensitivity and simplicity, it holds great potential in providing a promising platform for convenient and highly sensitive detection of PNK activity and its inhibitors.
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Label-free colorimetric assay for base excision repair Enzyme activity based on Nicking Enzyme assisted signal amplification.
Biosensors & bioelectronics, 2013Co-Authors: Xiaojuan Liu, Xiuzhong Wang, Ting Hou, Mingqin Chen, Shufeng LiuAbstract:Specific and sensitive detection of base excision repair Enzyme activity is essential to many fundamental biochemical process researches. Here, we propose a novel label-free homogeneous strategy for visualized uracil DNA glycosylase (UDG) activity assay based on Nicking Enzyme assisted signal amplification. In this method two hairpin probes were employed for the colorimetric detection, namely hairpin probe 1 (HP 1) carrying two uracil residues in the stem, and hairpin probe 2 (HP 2) containing a G-riched DNAzyme segment, and the recognition sequence as well as the cleavage site for the Nicking Enzyme. In the presence of UDG, the uracil bases in the stem of HP 1 can be specifically recognized and hydrolyzed by UDG, which leads to the destabilization of its stem containing abasic sites (AP sites), and then results in the opening of HP 1 to form a single strand. The opened HP 1 hybridizes with HP 2 to form a DNA duplex, which initiates the specific cleavage of HP 2 by the Nicking Enzyme, leading to the release of G-riched DNAzyme segments. As a result, HP 1 is released and able to hybridize with another HP 2 to induce the continuous cleavage of HP 2, generating enormous amount of G-riched DNAzyme segments. Finally, the G-riched DNAzyme segments bind hemin to form a catalytically active G-quadruplex–hemin DNAzyme which can catalyze the H2O2-mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−) to the colored ABTS–, providing a visible signal for UDG activity detection. This assay exhibits several advantages such as simplicity, low-cost, high selectivity and desirable sensitivity, which shows great potential of providing a promising platform for convenient and visualized analysis of UDG or other biomolecules.