催化发夹组装用于碱基切除修复酶活性检测研究
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摘要
碱基切除修复酶在DNA损伤修复过程中具有重要作用,且其检测与癌症等疾病的诊断相关。传统的碱基切除修复酶检测方法操作复杂、灵敏度低,且仅对酶的浓度进行定量分析而非酶的活性。为此,建立了基于催化发夹组装介导信号放大用于核酸内切酶IV(Endo IV)活性的检测方法。该方法利用Endo IV的活性作用于底物探针,将引发序列释放而引发催化发夹自组装信号放大,以实现Endo IV的活性检测分析。通过荧光检测实验可知,该方法检测下限为3.7×10-7 U/mL,可选择性地对Endo IV的活性进行检测,是一种设计简单、操作简便、灵敏度高的碱基切除修复酶活性检测方法。
Base excision repair enzymes played an important role in DNA damage repair, and their detection was related to cancer and disease diagnosis. The traditional detection of base excision repair enzymes was complex and lowsensitive, which only quantitatively analyzed the concentration of enzymes, rather than enzyme activity detection. A signal amplification platform based on catalytic hairpin self-assembly was established to detect endonuclease IV activity in base excision repair enzymes. This method was based on the activation of endonuclease IV acting on the substrate probe and releasing the initiation sequence, which caused the amplification of self-assembly signal of catalytic hairpin to realize the detection and analysis of endonuclease IV activity. According to the fluorescence detection experiment, the detection lower limit of this method was 3.7×10-7 U/mL, and Endo IV activity could be selectively detected. Therefore, this method was simple in design and operation and high in sensitivity.
Base excision repair enzymes played an important role in DNA damage repair, and their detection was related to cancer and disease diagnosis. The traditional detection of base excision repair enzymes was complex and lowsensitive, which only quantitatively analyzed the concentration of enzymes, rather than enzyme activity detection. A signal amplification platform based on catalytic hairpin self-assembly was established to detect endonuclease IV activity in base excision repair enzymes. This method was based on the activation of endonuclease IV acting on the substrate probe and releasing the initiation sequence, which caused the amplification of self-assembly signal of catalytic hairpin to realize the detection and analysis of endonuclease IV activity. According to the fluorescence detection experiment, the detection lower limit of this method was 3.7×10-7 U/mL, and Endo IV activity could be selectively detected. Therefore, this method was simple in design and operation and high in sensitivity.
引文
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[15]FLAENDER M,COSTA G,NONGLATON G,et al.A DNA Array Based on Clickable Lesion-Containing Hairpin Probes for Multiplexed Detection of Base Excision Repair Activities[J].Analyst,2016,141(22):6208-6216.
[15]FLAENDER M,COSTA G,NONGLATON G,et al.A DNA Array Based on Clickable Lesion-Containing Hairpin Probes for Multiplexed Detection of Base Excision Repair Activities[J].Analyst,2016,141(22):6208-6216.
[16]周礼义,龚亮,李学强,等.基于分子工程能量转移构建的比率型双光子荧光探针在生物成像中的应用[J].包装学报,2018,10(1):54-66.ZHOU Liyi,GONG Liang,LI Xueqiang,et al.Molecular Engineering of Energy Transfer-Based Ratiometric Two-Photon Fluorescent Probes for Bioimaging Applications[J].Packaging Journal,2018,10(1):54-66.
[2]WILSONIII D,BOHR V.The Mechanics of Base Excision Repair,and Its Relationship to Aging and Disease[J].DNA Repair,2007,6(4):544-559.
[3]MOL C D,IZUMI T,MITRA S,et al.DNA-Bound Structures and Mutants Reveal Abasic DNA Binding by APE1 DNA Repair and Coordination[J].Nature,2000,403(6768):451-456.
[4]LI Q,WANG Y D,SHEN G L,et al.Split Aptamer Mediated Endonuclease Amplification for Small-Molecule Detection[J].Chemical Communications,2015,51(20):4196-4199.
[5]LJUNGMAN M.Targeting the DNA Damage Response in Cancer[J].Chemical Reviews,2009,109(7):2929-2950.
[6]A B B O T T S R,M A D H U S U D A N S.H u m a n A PEndonuclease 1(APE1):From Mechanistic Insights to Druggable Target in Cancer[J].Cancer Treatment Reviews,2010,36(5):425-435.
[7]WANG D,LUO M,KELLEY M R.Human Apurinic Endonuclease 1(APE1)Expression and Prognostic Significance in Osteosarcoma:Enhanced Sensitivity of Osteosarcoma to DNA Damaging Agents Using Silencing RNA APE1 Expression Inhibition[J].Molecular Cancer Therapeutics,2004,3(6):679-686.
[8]WILSON III D M.Ape1 Abasic Endonuclease Activity is Regulated by Magnesium and Potassium Concentrations and is Robust on Alternative DNA Structures[J].Journal of Molecular Biology,2005,345(5):1003-1014.
[9]CESARATTO L,CODARIN E,VASCOTTO C,et al.Specific Inhibition of The Redox Activity of Ape1/Ref-1by E3330 Blocks TNF-α-Induced Activation of IL-8Production in Liver Cancer Cell Lines[J].PLoS One,2013,8(8):e70909.doi:10.1371/journal.pone.0070909.
[10]ZHUO Y,LIAO N,CHAI Y Q,et al.Ultrasensitive Apurinic/Apyrimidinic Endonuclease 1 Immunosensing Based on Self-Enhanced Electrochemiluminescence of a Ru(II)Complex[J].Analytical Chemistry,2014,86(2):1053-1060.
[11]HAN J,ZHUO Y,CHAI Y Q,et al.Ultrasensitive Electrochemical Strategy for Trace Detection of APE-1Via Triple Signal Amplification Strategy[J].Biosensors and Bioelectronics,2013,41:116-122.
[12]FANG S M,CHEN L,ZHAO M P.Unimolecular Chemically Modified DNA Fluorescent Probe for OneStep Quantitative Measurement of the Activity of Human Apurinic/Apyrimidinic Endonuclease 1 in Biological Samples[J].Analytical Chemistry,2015,87(24):11952-11956.
[13]ZHAI J Q,LIU Y B,HUANG S,et al.A Specific DNA-Nanoprobe for Tracking the Activities of Human Apurinic/Apyrimidinic Endonuclease 1 in Living Cells[J].Nucleic Acids Research,2017,45(6):e45.
[14]HUANG Y F,MA Y F,LI Y N,et al.Sensitive and Label-Free Fluorescence Detection of Apurinic/apyrimidinic Endonuclease 1 Activity Based on Isothermal Amplified-Generation of G-Quadruplex[J].New Journal of Chemistry,2017,41(5):1893-1896.
[15]FLAENDER M,COSTA G,NONGLATON G,et al.A DNA Array Based on Clickable Lesion-Containing Hairpin Probes for Multiplexed Detection of Base Excision Repair Activities[J].Analyst,2016,141(22):6208-6216.
[15]FLAENDER M,COSTA G,NONGLATON G,et al.A DNA Array Based on Clickable Lesion-Containing Hairpin Probes for Multiplexed Detection of Base Excision Repair Activities[J].Analyst,2016,141(22):6208-6216.
[16]周礼义,龚亮,李学强,等.基于分子工程能量转移构建的比率型双光子荧光探针在生物成像中的应用[J].包装学报,2018,10(1):54-66.ZHOU Liyi,GONG Liang,LI Xueqiang,et al.Molecular Engineering of Energy Transfer-Based Ratiometric Two-Photon Fluorescent Probes for Bioimaging Applications[J].Packaging Journal,2018,10(1):54-66.