Establishment Of Assay Methods For Tumor-associated DNA Repair Enzymes: HOGG1 And MTH1

Reactive oxygen species(ROS)are produced as a result of aerobic metabolism or exposure to external oxidative environment,which may oxidize DNA and precursor nucleotides to cause mutations.Guanines are most easily oxidized to 8-oxo-7,8-dihydroguanine(8-oxo G)and 8-oxo-2'-deoxyguanosine triphosphate(8-oxo-d GTP)because of low redox potential.The 8-oxo-d GTP can be inserted into DNA to form 8-oxo G.If not removed,the 8-oxo G may cause a G:C?T:A transversion during replication by pairing with adenine.The presence of such mutation is often found in the coding sequences of oncogenes and tumor suppressor genes.So,the increase of 8-oxo G abundance is associated with an elevated risk for cancers.The repair of 8-oxo G depends on human 8-oxoguanine DNA glycosylase 1(hOGG1)and human Mut T homolog 1(MTH1).Current studies have shown that mutations of hOGG1 and MTH1 genes are closely related to the development of the cancer,and the activity of them is essential for evaluating susceptibility and prognosis of cancers in clinical practice.Therefore,development of simple,rapid,sensitive and accurate method for hOGG1 and MTH1 has become the focus of cancer reach.Herein,methods for the assay of hOGG1 and MTH1,based on their inherent activities,have been respectively designed,which may provide new insights into the clinical detection of tumor-assosiated DNA repaire enzymes.1.Development of an electrochemical assay method for human 8-oxoguanine DNA glycosylase 1.hOGG1 is an important DNA repair enzyme that can excise the 8-oxo G from damaged DNA and is implicated in the aetiology of many kinds of cancers.Here,we report an electrochemical method to assess the activity of hOGG1 by using a carefully designed DNA probe,which contains an internal 8-oxo G site and an electrochemical active tag.In this method,hOGG1 can selectively recognize the 8-oxo G site and cleave the probe DNA,releasing a methylene blue(MB)-labeled signal fragment.The signal fragment can then hybridize with the E-DNA that has been previously immobilized on a gold electrode surface.Consequently,a sensitive electrochemical technique,square wave voltammetry,can be employed for the signal readout,thus characterizing the activity of hOGG1.This method can exhibit high sensitivity and low limit of detection(0.015 unit m L-1),indicating potential clinical application in the future.2.Establishment of a detection method for human Mut T homolog 1 based on DNA chain extension.MTH1,an oxidized purine nucleoside triphosphatase,can hydrolyze 8-oxo-d GTP,thus avoiding its subsequent incorporation into DNA or RNA,and inhibiting gene mutations and tumorigenesis.In this work,we propose a novel chain extension-based method to analysis MTH1.In our designed system,a electrochemical signal(MB)-modifed DNA fragment was synthesized as the DNA extension template,which is rich in adenines.In the extention process,8-oxo-d GTP,a substitute for deoxythymidine triphosphate(d TTP),can be inserted into DNA to mispair with adenine by DNA polymerase.The electrochemical signal on the template can thus been screened.However,in the present of MTH1,8-oxo-d GTPs are hydrolyzed,thus the primer is unable to extend.The signal-strand DNA template can then hybridize with the capture DNA on a gold electrode surface.The electrochemical signal values of the MB can be recorded,which can be used to establish the relationship between the signal and the activity of MTH1.This method has displayed excellent performance,indicating potential clinical application in the future.