CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deleti...CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease(T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.展开更多
An ultrasensitive electrochemical biosensor to detect trace Hg^(2+)in environmental samples was developed utilizing nanogold-decorated magnetic reduced graphene oxide(MrGO-AuNPs),exonuclease III-assisted target cycle(...An ultrasensitive electrochemical biosensor to detect trace Hg^(2+)in environmental samples was developed utilizing nanogold-decorated magnetic reduced graphene oxide(MrGO-AuNPs),exonuclease III-assisted target cycle(Exo Ⅲ-ATC)and hybridization chain reaction(HCR)synergistic triple signal amplification.The MrGO-AuNPs is a superior carrier for capture DNA(cDNA)and acts as magnetic media for automatic separation and adsorption.This innovative utilization of the magnetism and improved sensing efficiency obviates the need for direct modification and repeated polishing of the working electrode.Additionally,the three DNA hairpins(cDNA,methylene blue(MB)labeled HP1 and HP2)further contribute to biosensor specificity and selectivity.When cDNA captures Hgt,it activates Exo Ⅲ-ATC due to the formation of a sticky end in the DNA stem via thymine-Hig-thymidine(T-Hg^(2+)-T),this leads to the hydrolysis of self-folded DNA by Exo Ⅲ-ATC to form"key"DNA(kDNA).The kDNA subsequently initiates HCR,resulting in massive super-sandwich structures(kDNA-[HP1/HP2])carrying signaling molecules on MrGO-AuNPs,and this overall structure serves as a signal probe(SP).Leveraging magnetic adsorption,the SP was automatically adsorbed onto the magneto-glass carbon electrode(MGCE),generating an amplified signal.This biosensor's detection limit(LOD)was 3.14 pmol/L,far below the limit of 10 nmol/L for mercury in drinking water set by the US EPA.The biosensor also showed excellent selectivity when challenged by interfering ions,and the results of its application in actual samples indicate that it has good potential for practical applications in environmental monitoring.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
In plants and mammals,non-homologous end-joining is the dominant pathway to repair DNA doublestrand breaks,making it challenging to generate knock-in events.In this study,we identified two groups of exonucleases from ...In plants and mammals,non-homologous end-joining is the dominant pathway to repair DNA doublestrand breaks,making it challenging to generate knock-in events.In this study,we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana.We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems.In Arabidopsis thaliana,fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants.In addition,we demonstrated stable and heritable knock-ins in wheat in 1%of the primary transformants.Taken together,our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.展开更多
I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of canc...I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have dis- covered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases I, III and IV. I discovered the mam- malian exonucleases DNase III （TREX1） and IV （FEN1）. Interestingly, expression of TREXI was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O6-methylguanine （O6mG） is repaired without removing the guanine from DNA, identifying a sur- prising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation.展开更多
Prime editing(PE)is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates,but is limited by low editing efficiency.We previously fused the M-MLV reverse transcriptase t...Prime editing(PE)is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates,but is limited by low editing efficiency.We previously fused the M-MLV reverse transcriptase to the Cas9 nickase,generating the PE2(v1)system,but the editing efficiency of this system is still low.Here we develop different versions of PE2 by adding the 50-to-30 exonuclease at different positions of the nCas9-M-MLV RT fusion protein.PE2(v2),in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions,deletions,and insertions at several genomic sites by 1.7-to 2.9-fold in plant cells compared to PE2(v1).The improved editing efficiency of PE2(v2)is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1,PE-P2,and PPE systems.Using PE2(v2),we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites.The PE efficiency is further improved by combining PE2(v2)and dualpegRNAs.Taken together,the increased genome editing efficiency of PE2(v2)developed in this study may enhance the applications of PE in plants.展开更多
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
Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)...Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)variants of PARN-like ribonuclease domain-containing exonuclease 1(PNLDC1)have been reported to experience infertility with nonobstructive azoospermia.The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia(OAT)in a patient from a Chinese Han family.Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant(NM_173516.2,c.l42C>T,p.Gln48Ter)in PNLDC1.Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype,including microcephaly,head tapering,and globozoospermia.Consistently,peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome.Furthermore,the disomy rate of chromosomes in the patient’s spermatozoa was significantly increased compared with that of a fertile control sample.We reported an LOF variant of the PNLDC1 gene responsible for OAT.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
Detection of miRNAs presents a particular challenge because of their limited size, high sequence homo- logy and greatly various expression level. In this work, an ultrasensitive, label-free and isothermal miRNA detect...Detection of miRNAs presents a particular challenge because of their limited size, high sequence homo- logy and greatly various expression level. In this work, an ultrasensitive, label-free and isothermal miRNA detection was developed based on asymmertric hairpin probe, exonuclease I（Exo I） and SYBR Green I. The method employed asymmetric hairpin probe to perform cycled polymerization and Exo I to reduce background signal. In the presence of the target miRNA, the target triggers probe-mediated cycled polymerization reactions to generate lots of dsDNA products. The dsDNA product effectively prevents itself from being degraded by Exo I and permitted the insertion of more fluorescence dye into it to enlarge the fluorescence signal. In the absence of the target miRNA, there was no probe-mediated polymerization reaction, and the probe was digested by Exo I added, which minimized the intercala- tion of fluorescence dye to reduce the background signal. The combination of the probe-mediated cycled polymeriza- tion with the Exo 1-assisted background reduction allows us to achieve a detection limit of 5× 10^-18 mol/L. Owing to its ultrasensitivity, excellent specificity, convenience and low-cost, this method might hold out great promise in miRNA detection.展开更多
基金supported by grants from the National Transgenic Science and Technology Program of China(2019ZX08010-003,2019ZX08010-001,2018ZX0801002B)the National Key Research and Development Program of China(2016YFD0100602)+1 种基金the Scientific Program of Beijing Municipal Commission of Science and Technology(Z171100001517001)the National Natural Science Foundation of China(31672015)to J.L.Q。
文摘CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease(T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.
基金This study was supported by the Key Research and Development Program of China(No.2018YFC0213400)the National Natural Science Foundation of China(Nos.21737002,21976119,and 22176126)。
文摘An ultrasensitive electrochemical biosensor to detect trace Hg^(2+)in environmental samples was developed utilizing nanogold-decorated magnetic reduced graphene oxide(MrGO-AuNPs),exonuclease III-assisted target cycle(Exo Ⅲ-ATC)and hybridization chain reaction(HCR)synergistic triple signal amplification.The MrGO-AuNPs is a superior carrier for capture DNA(cDNA)and acts as magnetic media for automatic separation and adsorption.This innovative utilization of the magnetism and improved sensing efficiency obviates the need for direct modification and repeated polishing of the working electrode.Additionally,the three DNA hairpins(cDNA,methylene blue(MB)labeled HP1 and HP2)further contribute to biosensor specificity and selectivity.When cDNA captures Hgt,it activates Exo Ⅲ-ATC due to the formation of a sticky end in the DNA stem via thymine-Hig-thymidine(T-Hg^(2+)-T),this leads to the hydrolysis of self-folded DNA by Exo Ⅲ-ATC to form"key"DNA(kDNA).The kDNA subsequently initiates HCR,resulting in massive super-sandwich structures(kDNA-[HP1/HP2])carrying signaling molecules on MrGO-AuNPs,and this overall structure serves as a signal probe(SP).Leveraging magnetic adsorption,the SP was automatically adsorbed onto the magneto-glass carbon electrode(MGCE),generating an amplified signal.This biosensor's detection limit(LOD)was 3.14 pmol/L,far below the limit of 10 nmol/L for mercury in drinking water set by the US EPA.The biosensor also showed excellent selectivity when challenged by interfering ions,and the results of its application in actual samples indicate that it has good potential for practical applications in environmental monitoring.
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funded by grant no.031B0548 in the frame of the program"Crop plants of the future"from the Bundesministerium fur Bildung und Forschung to A.T.funded by the Investissement d’Avenir program of the French National Agency of Research for the project GENIUS(ANR-11-BTBR-0001_GENIUS).
文摘In plants and mammals,non-homologous end-joining is the dominant pathway to repair DNA doublestrand breaks,making it challenging to generate knock-in events.In this study,we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana.We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems.In Arabidopsis thaliana,fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants.In addition,we demonstrated stable and heritable knock-ins in wheat in 1%of the primary transformants.Taken together,our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.
文摘I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have dis- covered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases I, III and IV. I discovered the mam- malian exonucleases DNase III （TREX1） and IV （FEN1）. Interestingly, expression of TREXI was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O6-methylguanine （O6mG） is repaired without removing the guanine from DNA, identifying a sur- prising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation.
基金supported by grants from the National Key Research and Development Program of China(2022YFF1002802)the National Natural Science Foundation of China(32170410)the Science and Technology Innovation Young Talent Team of Shanxi Province(202204051001019).
文摘Prime editing(PE)is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates,but is limited by low editing efficiency.We previously fused the M-MLV reverse transcriptase to the Cas9 nickase,generating the PE2(v1)system,but the editing efficiency of this system is still low.Here we develop different versions of PE2 by adding the 50-to-30 exonuclease at different positions of the nCas9-M-MLV RT fusion protein.PE2(v2),in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions,deletions,and insertions at several genomic sites by 1.7-to 2.9-fold in plant cells compared to PE2(v1).The improved editing efficiency of PE2(v2)is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1,PE-P2,and PPE systems.Using PE2(v2),we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites.The PE efficiency is further improved by combining PE2(v2)and dualpegRNAs.Taken together,the increased genome editing efficiency of PE2(v2)developed in this study may enhance the applications of PE in plants.
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 Key Research and Development Program of China(2022YFC2702604)the National Natural Science Foundation of China(82171608,82201773,and 81971447)+1 种基金the China Postdoctoral Science Foundation(2022M711119)the Scientific Research Foundation of the Health Committee of Hunan Province(B202301039323 and B202301039518).
文摘Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)variants of PARN-like ribonuclease domain-containing exonuclease 1(PNLDC1)have been reported to experience infertility with nonobstructive azoospermia.The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia(OAT)in a patient from a Chinese Han family.Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant(NM_173516.2,c.l42C>T,p.Gln48Ter)in PNLDC1.Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype,including microcephaly,head tapering,and globozoospermia.Consistently,peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome.Furthermore,the disomy rate of chromosomes in the patient’s spermatozoa was significantly increased compared with that of a fertile control sample.We reported an LOF variant of the PNLDC1 gene responsible for OAT.
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.
基金Supported by the National Natural Science Foundation of China（No.21275043） and the National Basic Research Program of China（No .2009CB421601）.
文摘Detection of miRNAs presents a particular challenge because of their limited size, high sequence homo- logy and greatly various expression level. In this work, an ultrasensitive, label-free and isothermal miRNA detection was developed based on asymmertric hairpin probe, exonuclease I（Exo I） and SYBR Green I. The method employed asymmetric hairpin probe to perform cycled polymerization and Exo I to reduce background signal. In the presence of the target miRNA, the target triggers probe-mediated cycled polymerization reactions to generate lots of dsDNA products. The dsDNA product effectively prevents itself from being degraded by Exo I and permitted the insertion of more fluorescence dye into it to enlarge the fluorescence signal. In the absence of the target miRNA, there was no probe-mediated polymerization reaction, and the probe was digested by Exo I added, which minimized the intercala- tion of fluorescence dye to reduce the background signal. The combination of the probe-mediated cycled polymeriza- tion with the Exo 1-assisted background reduction allows us to achieve a detection limit of 5× 10^-18 mol/L. Owing to its ultrasensitivity, excellent specificity, convenience and low-cost, this method might hold out great promise in miRNA detection.
