基于PFP荧光共振能量转移原理检测氧化损伤DNA
|
摘要
提出一种基于水溶性共轭聚合物PFP荧光共振能量转移原理检测氧化损伤DNA的方法。通过加入DNA修复酶来识别并切除被氧化的DNA碱基,获得含磷酸基团核苷酸空隙的DNA,再通过羟基引入荧光标记物,加入PFP后可得到PFPDNA复合物,通过荧光共振能量转移(fluorescence resonance energy transfer,FRET)进行氧化损伤DNA的检测。对方法的影响因素进行了考查和优化,包括PFP的浓度、DNA修复酶的种类、DNA聚合酶I的用量以及芬顿反应的时间等。研究表明,基于PFP荧光共振能量转移原理检测氧化损伤DNA方法具有灵敏度高和特异性强的优势,可确保检测的准确性,对老年疾病的预防医学具有很好的应用前景。
A fluorescence resonance energy transfer based on water-soluble conjugated polymer PFP is proposed to detect oxidative damage to DNA. This method can identify and excise the oxidized DNA base by adding DNA repair enzyme to obtain DNA containing 3' →5' phosphoric acid group nucleotides gap,introduce hydroxyl group at3'→5' site,and then introduce the fluorescent marker. After adding PFP,the PFP-DNA complex can be obtained.Fluorescence resonance energy transfer( FRET) was used to detect DNA in oxidative damage. The influence factors of this method were investigated and optimized,including the concentration of PFP,the type of DNA repair enzyme,the concentration of DNA polymerase I and the time of Fenton reaction. The study shows that the detection method of oxidative damage DNA based on PFP fluorescence resonance energy transfer principle has the advantages of high sensitivity and specificity,which can ensure the accuracy of detection,and has a good application prospect for the preventive medicine of senile diseases.
A fluorescence resonance energy transfer based on water-soluble conjugated polymer PFP is proposed to detect oxidative damage to DNA. This method can identify and excise the oxidized DNA base by adding DNA repair enzyme to obtain DNA containing 3' →5' phosphoric acid group nucleotides gap,introduce hydroxyl group at3'→5' site,and then introduce the fluorescent marker. After adding PFP,the PFP-DNA complex can be obtained.Fluorescence resonance energy transfer( FRET) was used to detect DNA in oxidative damage. The influence factors of this method were investigated and optimized,including the concentration of PFP,the type of DNA repair enzyme,the concentration of DNA polymerase I and the time of Fenton reaction. The study shows that the detection method of oxidative damage DNA based on PFP fluorescence resonance energy transfer principle has the advantages of high sensitivity and specificity,which can ensure the accuracy of detection,and has a good application prospect for the preventive medicine of senile diseases.
引文
1 Kowalczyk S W,Hall A R,Dekker C.Detection of local protein structures along DNA using solid-state nanopores.Nano Letters,2010;10(1):324-328
2 Lovell M A,Markesbery W R.Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer's disease.Nucleic Acids Research,2007;35(22):7497-7504
3 Debalsi K L,Hoff K E,Copeland W C.Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis,aging and age-related diseases.Ageing Research Reviews,2017;33:89-104
4于晨,董超然,张照辉,等.8-羟基脱氧鸟嘌呤作为DNA氧化损伤标志物的研究现状.中国临床药理学杂志,2017;33(13):1267-1270Yu Chen,Dong Chaoran,Zhang Zhaohui,et al.Review of the research on 8-hydroxy-2 deoxyguanosine as a DNA oxidative damage marker.The Chinese Journal of Clinical Pharmacology,2017;33(13):52-56
5 Imlay J A.The molecular mechanisms and physiological consequences of oxidative stress:Lessons from a model bacterium.Nature Reviews Microbiology,2013;11(7):443-454
6 Santos R X,Correia S C,Zhu X,et al.Mitochondrial DNA oxidative damage and repair in aging and Alzheimer's disease.Antioxidants&Redox Signaling,2013;18(18):2444-2457
7 Friedman J I,Stivers J T.Detection of damaged DNA bases by DNAglycosylase enzymes.Biochemistry,2010;49(24):4957-4967
8 Karlsson H L.The comet assay in nanotoxicology research.Analytical and Bioanalytical Chemistry,2010;398(2):651-666
9 Azqueta A,Collins A R.The essential comet assay:A comprehensive guide to measuring DNA damage and repair.Archives of Toxicology,2013;87(6):949-968
10 Cadet J,Douki T,Ravanat J L.Oxidatively generated base damage to cellular DNA.Free Radical Biology and Medicine,2010;49(1):9-21
11 Liu M,Bandaru V,Bond J P,et al.The mouse ortholog of NEIL3is a functional DNA glycosylase in vitro and in vivo.Proceedings of the National Academy of Sciences,2010;107(11):4925-4930
12 Phillips D H,Arlt V M.The32P-postlabeling assay for DNA adducts.Nature Protocols,2007;2(11):2772-2781
13 Gan W,Nie B,Shi F,et al.Age-dependent increases in the oxidative damage of DNA,RNA,and their metabolites in normal and senescence-accelerated mice analyzed by LC-MS/MS:Urinary 8-oxoguanosine as a novel biomarker of aging.Free Radical Biology and Medicine,2012;52(9):1700-1707
14 Dinckaya E,Kinik O,Sezgintürk M K,et al.Development of an impedimetric aflatoxin M1 biosensor based on a DNA probe and gold nanoparticles.Biosensors and Bioelectronics,2011;26(9):3806-3811
15 Wu Y,Zhang B,Guo L H.Label-free and selective photoelectrochemical detection of chemical DNA methylation damage using DNArepair enzymes.Analytical Chemistry,2013;85(14):6908-6914
16陈逸青,刘从云,陈学全,等.淫羊藿总黄酮对丝裂霉素致小鼠骨髓细胞DNA损伤的保护作用.毒理学杂志,2010;24(3):222-224Chen Yiqing,Liu Congyun,Chen Xuequan,et al.Protective effects of Epimedium Flavonoids on DNA damage induced by mitomycin Cin bone marrow cells of mice.Journal of Toxicology,2010;24(3):222-224
17 Rogers K R,Apostol A,Steen J M,et al.Fiber optic biosensor for detection of DNA damage.Analytica Chimica Acta,2001;444(1):51-60
18 Li C,Wang H.Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases.Journal of Chromatography A,2015;1406:324-330
2 Lovell M A,Markesbery W R.Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer's disease.Nucleic Acids Research,2007;35(22):7497-7504
3 Debalsi K L,Hoff K E,Copeland W C.Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis,aging and age-related diseases.Ageing Research Reviews,2017;33:89-104
4于晨,董超然,张照辉,等.8-羟基脱氧鸟嘌呤作为DNA氧化损伤标志物的研究现状.中国临床药理学杂志,2017;33(13):1267-1270Yu Chen,Dong Chaoran,Zhang Zhaohui,et al.Review of the research on 8-hydroxy-2 deoxyguanosine as a DNA oxidative damage marker.The Chinese Journal of Clinical Pharmacology,2017;33(13):52-56
5 Imlay J A.The molecular mechanisms and physiological consequences of oxidative stress:Lessons from a model bacterium.Nature Reviews Microbiology,2013;11(7):443-454
6 Santos R X,Correia S C,Zhu X,et al.Mitochondrial DNA oxidative damage and repair in aging and Alzheimer's disease.Antioxidants&Redox Signaling,2013;18(18):2444-2457
7 Friedman J I,Stivers J T.Detection of damaged DNA bases by DNAglycosylase enzymes.Biochemistry,2010;49(24):4957-4967
8 Karlsson H L.The comet assay in nanotoxicology research.Analytical and Bioanalytical Chemistry,2010;398(2):651-666
9 Azqueta A,Collins A R.The essential comet assay:A comprehensive guide to measuring DNA damage and repair.Archives of Toxicology,2013;87(6):949-968
10 Cadet J,Douki T,Ravanat J L.Oxidatively generated base damage to cellular DNA.Free Radical Biology and Medicine,2010;49(1):9-21
11 Liu M,Bandaru V,Bond J P,et al.The mouse ortholog of NEIL3is a functional DNA glycosylase in vitro and in vivo.Proceedings of the National Academy of Sciences,2010;107(11):4925-4930
12 Phillips D H,Arlt V M.The32P-postlabeling assay for DNA adducts.Nature Protocols,2007;2(11):2772-2781
13 Gan W,Nie B,Shi F,et al.Age-dependent increases in the oxidative damage of DNA,RNA,and their metabolites in normal and senescence-accelerated mice analyzed by LC-MS/MS:Urinary 8-oxoguanosine as a novel biomarker of aging.Free Radical Biology and Medicine,2012;52(9):1700-1707
14 Dinckaya E,Kinik O,Sezgintürk M K,et al.Development of an impedimetric aflatoxin M1 biosensor based on a DNA probe and gold nanoparticles.Biosensors and Bioelectronics,2011;26(9):3806-3811
15 Wu Y,Zhang B,Guo L H.Label-free and selective photoelectrochemical detection of chemical DNA methylation damage using DNArepair enzymes.Analytical Chemistry,2013;85(14):6908-6914
16陈逸青,刘从云,陈学全,等.淫羊藿总黄酮对丝裂霉素致小鼠骨髓细胞DNA损伤的保护作用.毒理学杂志,2010;24(3):222-224Chen Yiqing,Liu Congyun,Chen Xuequan,et al.Protective effects of Epimedium Flavonoids on DNA damage induced by mitomycin Cin bone marrow cells of mice.Journal of Toxicology,2010;24(3):222-224
17 Rogers K R,Apostol A,Steen J M,et al.Fiber optic biosensor for detection of DNA damage.Analytica Chimica Acta,2001;444(1):51-60
18 Li C,Wang H.Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases.Journal of Chromatography A,2015;1406:324-330