Monitoring telomerase activity with high sensitive and reliable is of great importance to cancer analysis. In this paper, we report a sensitive and facile method to detect telomerase activity using AIEgens mod- ified ...Monitoring telomerase activity with high sensitive and reliable is of great importance to cancer analysis. In this paper, we report a sensitive and facile method to detect telomerase activity using AIEgens mod- ified probe （TPE-Py-DNA） as a fluorescence reporter and exonuclease llI （Exo lIl） as a signal amplifier. With the aid of telomerase, repeat units （TrAGGG）n are extended from the end of template substrate oligonucleotides （TS primer） that form duplex DNAs with TPE-Py-DNA. Then, Exo llI catalyzes the diges- tion of duplex DNAs, liberating elongation product and releasing hydrophobic TPE-Py. The released hydrophobic TPE-Py aggregate together and produce a telomerase-activity-related fluorescence signal. The liberated product hybridizes with another TPE-Py-DNA probe, starting the second cycle. Finally, we obtain the target-to-signal amplification ratio of 1 ：N2. This strategy exhibits good performance for detecting clinical urine samples （distinguishing 15 cancer patients＇ samples from 8 healthy ones） and checking intracellular telomerase activity （differentiating cell lines including HeLa, MDA-MB-231, MCF-7, A375, HLF and MRC-5 from the cells pretreated with telomerase-related drug）, which shows its potential in clinical diagnosis as well as therapeutic monitoring of cancer.展开更多
The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-...The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.展开更多
Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly fo...Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly formation of a trivalent G-quadruplex/hemin DNAzyme for colorimetric detection of Hg^(2+).A hairpin DNA(Hr)was designed with thymine-Hg^(2+)-thymine pairs that catalyzed by Exo III is prompted to happen upon binding Hg^(2+).A released DNA fragment triggers the catalytic assembly of other three hairpins(H1,H2,and H3)to form many trivalent G-quadruplex/hemin DNA enzymes for signal output.The developed sensor shows a dynamic range from 2 pM to 2μM,with an impressively low detection limit of 0.32 pM for Hg^(2+)detection.Such a sensor also has good selectivity toward Hg^(2+)detection in the presence of other common metal ions.This strategy shows the great potential for visual detection with portable type.展开更多
A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A doub...A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) to generate TCDD:AhR:DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.展开更多
基金supported by the National Natural Science Foundation of China(21375042,21405054,21525523,21574048,and21404028)the National Basic Research Program of China(2015CB932600,2013CB933000,and 2016YFF0100800)+1 种基金the Special Fund for Strategic New Industry Development of Shenzhen,China(JCYJ20150616144425376)1000 Young Talent Program(to F.Xia)
文摘Monitoring telomerase activity with high sensitive and reliable is of great importance to cancer analysis. In this paper, we report a sensitive and facile method to detect telomerase activity using AIEgens mod- ified probe （TPE-Py-DNA） as a fluorescence reporter and exonuclease llI （Exo lIl） as a signal amplifier. With the aid of telomerase, repeat units （TrAGGG）n are extended from the end of template substrate oligonucleotides （TS primer） that form duplex DNAs with TPE-Py-DNA. Then, Exo llI catalyzes the diges- tion of duplex DNAs, liberating elongation product and releasing hydrophobic TPE-Py. The released hydrophobic TPE-Py aggregate together and produce a telomerase-activity-related fluorescence signal. The liberated product hybridizes with another TPE-Py-DNA probe, starting the second cycle. Finally, we obtain the target-to-signal amplification ratio of 1 ：N2. This strategy exhibits good performance for detecting clinical urine samples （distinguishing 15 cancer patients＇ samples from 8 healthy ones） and checking intracellular telomerase activity （differentiating cell lines including HeLa, MDA-MB-231, MCF-7, A375, HLF and MRC-5 from the cells pretreated with telomerase-related drug）, which shows its potential in clinical diagnosis as well as therapeutic monitoring of cancer.
基金supported by grants from the National Natural Science Foundation of China (No.21472060)Shenzhen Municipal government (Nos.JCYJ20160301153959476 and JCYJ20160324163734374)Shenzhen Reform Commission (Disciplinary Development Program for Chemical Biology)
文摘The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.
基金Supported by The Science and Technology Project of General Administration of Quality Supervision,Inspection and Quarantine (2015IK126)The Science and Technology Project of Changsha City of Hunan Province of China (KQ1602124).
文摘Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly formation of a trivalent G-quadruplex/hemin DNAzyme for colorimetric detection of Hg^(2+).A hairpin DNA(Hr)was designed with thymine-Hg^(2+)-thymine pairs that catalyzed by Exo III is prompted to happen upon binding Hg^(2+).A released DNA fragment triggers the catalytic assembly of other three hairpins(H1,H2,and H3)to form many trivalent G-quadruplex/hemin DNA enzymes for signal output.The developed sensor shows a dynamic range from 2 pM to 2μM,with an impressively low detection limit of 0.32 pM for Hg^(2+)detection.Such a sensor also has good selectivity toward Hg^(2+)detection in the presence of other common metal ions.This strategy shows the great potential for visual detection with portable type.
基金This project was supported by grants from National Natu ral Science Foundation of China (No. 20107002,20377017).
文摘A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) to generate TCDD:AhR:DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.
