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  • br Conclusions In summary we have designed an

    2021-10-09


    Conclusions In summary, we have designed an ultrasensitive electrochemical assay for hOGG1 based on a ratiometric strategy and HCR assisted amplification. The hOGG1 can specifically identify 8-oxo G site on the DNA probes modified on the electrode surface. The subsequent cleavage reaction triggers HCR and leads to the changes of electrochemical signals of Fc and [Ru(NH3)6]3+. After analyzing the electrochemical responses, ultrasensitive detection of hOGG1 is achieved. The low LOD is owning to the employment of two electrochemical probes and HCR assisted signal amplification. In addition, the whole assay avoids the utilization of any kind of enzymatic reactions for amplification, thus the operation is pretty simple with low cost. Based the above merits, this method might hold great potential for further applications in bioanalysis, early clinical diagnosis and biomedical researches.
    Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant no. 81801792), the Key R&D Program of Tianjin (Grant no. 18YFZCSY01290), the Scientific Research Instrument Developing Project of the Chinese Academy of Sciences (Grant no. YJKYYQ20170067) and the Science and Technology Program of Suzhou (Grant no. SS201867).
    Introduction DNA glycosylase proteins are involved in 5416 excision repair and catalyze the repair process by cleaving the N-glycosidic bond of damaged or mispaired nucleotide [1]. MUTYH, one of the several members of the DNA glycosylase family, is the gene responsible for MUTYH-associated polyposis (MAP), a hereditary disease characterized by colorectal polyposis and carcinoma(s), and individuals with biallelic germline mutations of MUTYH are susceptible to MAP [2], [3]. Recently, NTHL1-associated polyposis (NAP) has been reported as another hereditary disease caused by germline mutations of a DNA glycosylase gene [4], [5], [6]. Individuals with biallelic germline mutations of the DNA glycosylase gene NTHL1 are susceptible to NAP, which is characterized by colorectal polyposis and multiple types of carcinomas, including colorectal carcinoma [4], [5], [6]. So far, many types of pathogenic MUTYH variants have been identified through analysis of genotype-phenotype correlations and functional analyses (e.g., DNA cleavage activity assay and mutation assay) of variant proteins [7], [8], [9], [10]. However, no proper functional evaluation of recombinant NTHL1 variant proteins found in NAP families has been performed yet. Damaged bases such as thymine glycol (Tg), 5-hydroxyuracil (5OHU), 5-formyluracil, 5-hydroxycytosine, 5-hydroxy-6-hydrothymine, 5,6-dihydroxycytosine, 5,6-dihydrouracil (DHU), and formamidopyrimidine have been reported as substrates of NTHL1 [11], [12], [13], [14]. To evaluate the repair capacity of NTHL1 variants, it is preferable to assess their ability to suppress damaged base-induced mutations as well as activity to excise damaged bases from DNA. However, little is known about the mutagenicity of the above-mentioned bases in human cells, with the exception that 5-formyluracil has been shown to be mutagenic, although weakly so, in human cells [15]. Thus, at present, no damaged bases are known to be suitable for use in the evaluation of NTHL1 variants’ ability to suppress mutations caused by damaged bases. Therefore, in this paper, we report our first attempt to examine the mutagenic property of 5OHU; one of the substrates of NTHL1. 5OHU is produced by oxidative de-amination of cytosine by reactive oxygen species [16]. Among the several types of oxidation products of cytosines, 5OHU has been reported to be abundantly produced [17], [18], [19]. Interestingly, 5OHU has been shown to form stable base-pairs with A, G, C and T residues in a DNA duplex, based on NMR spectroscopic and UV melting experiments [20], although there were some differences in the degree of stability; 5OHU formed the most stable pair with G, and least stable pair with C [21]. These data suggest the potential mutagenicity of 5OHU. With regard to human DNA polymerases bypassing 5OHU, human Polβ and Polι have been shown in an in vitro replication assay to preferentially incorporate A and T, respectively, opposite 5OHU [22]. While several other human DNA polymerases are known to exist [23], there have been no reports describing the ability of human DNA polymerases other than Polβ and Polι to bypass 5OHU. So far, the types of mutations that are most predominantly induced by 5OHU in human cells remain uncertain, although it has been shown to cause C→T mutation in Escherichia coli (E. coli) [24]. With regard to the DNA glycosylase proteins capable of excising 5OHU from DNA, NTHL1 and three other DNA glycosylases (SMUG1, NEIL1, and UNG2) have been reported [12], [25], [26], [27], [28]. However, the activity of the other DNA glycosylases for the excision of 5OHU remains unclear.