A dual enzymatic amplified strategy for the detection of endonuclease V activityby Wei Wang, Feifei Peng, Wang Li, Yan Huang, Zhou Nie, Shouzhuo Yao

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W. Li, Y. Huang, Z. Nie and S. Yao, Anal. Methods, 2015, DOI: 10.1039/C5AY01776A. 1

A Dual Enzymatic Amplified Strategy for the Detection of

Endonuclease V Activity

Wei Wang, Feifei Peng, Wang Li,* Yan Huang,* Zhou Nie, Shouzhuo Yao

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China.

Fax: +86-731-88821848; Tel: +86-731-88821626;

E-mail: wli@hnu.edu.cn, huangyan.hnu@gmail.com


Endonuclease V (EndoV) plays important roles in DNA repair. For the absence of the quantitative assay method for EndoV, we have developed a dual enzymatic amplified strategy for the detection of EndoV activity based on a nicking enzyme and a template independent polymerase. Every hydrolysis to substrate by EndoV can generate only one 3'-hydroxyl terminal to support the polymerization of polymerase, however, with the assistance of nicking enzyme, abundant 3'-hydroxyl terminals are generated. Next, terminal deoxynucleotidyl transferase (TdT) involved in the second amplified procedure prolongs the 3'-hydroxyl terminus DNA with repeated T bases, providing the long template for the synthesis of fluorescent CuNPs. Consequently, a wide linear dynamic range of 0.02 to 10 U/mL is reached with a detection limit of 0.02 U/mL. This method exhibits several advantages such as high sensitivity and desirable selectivity, which shows great potential of promising platform for sensitive analysis of EndoV or other biomolecules.


Single bases in DNA can be damaged by a variety of chemical reactions or optical radiation, and deamination of DNA bases is a common lesion caused by endogenous and environmental agents.1,2 The transformation of the amino to keto alters the hydrogen bonding properties of the damaged bases from a donor of hydrogen bond to an acceptor.1 Such nitrosative deamination of DNA bases can cause transition mutations during DNA replication.3 One of the repair pathways to remove the deaminated lesions referred to as nucleotide excision repair (NER). This type of pathway is initiated by a specific endonuclease that recognizes the damaged DNA and nicks the phosphodiester bond adjacent to the lesion site.4 Endonuclease V (EndoV), a specific NER endonuclease, recognizes deoxyinosine, a deamination product of deoxyadenosine in DNA.4,5 The hypoxanthine mispair with cytosine will generate an

A/T to G/C mutation,6 and cause cancer predispositions. The assay of EndoV activities represents a critical step toward the understanding of DNA damage repairing.

Therefore, developing new methods for EndoV assay is of fundamental importance, providing useful tools in the biological research, pathological study and target drug screening.

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DOI: 10.1039/C5AY01776A 2

Most of the studies on EndoV, however, is mainly concerning the qualitative analysis and mechanism study,1,3,7-10 there are rare works on its quantitative assay.

Moreover, these studies on EndoV is focus on the radioactive labeling and gel electrophoresis, which is indirect, unsafe and insensitive. Therefore, it is a great challenge to develop a convenient and efficient biosensor platform for the quantitative

EndoV assay.

Compared with other techniques, fluorescent analysis is a main approach to structure biosensors for its high sensitivity and simple manipulation. As an ideal alternative to conventional fluorophores such as organic dyes and quantum dots, fluorescent metal nanoparticles11-13 with intensive fluorescence emission have emerged as promising fluorescent probes. Recently, DNA-templated fluorescent copper nanoparticles (CuNPs) have drawn considerable attention for several advantages such as, low toxicity, good biocompatibility, large MegaStokes shifting, and its facile and quick synthesis procedure.13-15 One kind of DNA-templated CuNPs is using single-stranded (ssDNA) poly(thymine) (polyT) as template, and the fluorescent intensity of polyT-CuNPs is strictly depended on the length of single-stranded polyT.13,15 However, the commercial synthesis of long polyT ssDNA (hundreds of thymine bases) is complicated and costly, encouraging us to ponder on a non-commercial and in-situ synthesis method of long single-stranded polyT. In addition, to improve the sensitivity of the assay, nanoparticles and enzymes are usually employed to amplify signal.16-17 For the cumbersome procedures of synthesis and modification will complicate the usage of nanoparticles, to take the advantage of fascinating characteristics of enzymes, many researches have been focus on using hydrolytic enzymes and polymerases to develop signal amplification detect platform to improve the sensitivity.17-19