| DNA methylation, a crucial epigenetic modification of the genome, plays a significant role in both prokaryotes and eukaryotes,Indeed, aberrant DNA methylation is responsible to various diseases such as cancers. It is well known that DNA methylation is caused by DNA methyltransfease(MTase), which is able to transfer a methyl group from the methyl group donor of S-adenosylmethionine(SAM) to the C5-positon of cytosine or the 6-amino group of adenine at specific base sequence. Therefore, the DNA methylation level is always linked to MTase activity. And it is important to analyze the activity of DNA MTase and screen the corresponding inhibitors. There are many traditional methods for analyzing DNA MTase activity and screening inhibitor, which are focused on radioisotope labeled substrate, bisulfate treatment, colorimetric approaches, HPLC(high-performance liquid chromatography), and so on. However, all of them have limitations, for example, radioisotope labeled substrate are harmful to biological tissue, bisulfate treatment need to convert cytosine to uracil which is time-consuming. Although colorimetric methods are simple, gold nanoparticles are easy to aggregate. HPLC need sophisticated large-scale instrument. Thus, it is still necessary to develop rapid and simple methods for accurately detecting DNA methylation level and analyzing MTase activity. 5-hmC is often considered to be the sixth base of the genome, 5-hmC in mammals is tissue specific and non-random, which is relatively abundant in neuron cells, mouse cerebellum, and embryonic stem cells. Moreover, some studies present that the 5-hmC levels are profoundly reduced in many types of cancer cells, which indicates that the change of the expression level of 5-hmC may be used as biomarker for cancer diagnosis. there are various methods have been developed for detection of 5-hmC in genomic DNA such as, thin layer chromatography(TLC), enzymatic radioactive glycosylation labelling, and high-performance liquid chromatography(HPLC) with UV detection. However, though these methods have their own advantages, all of them have limitations still. For example, HPLC require expensive and sophisticated large-scale instrument.Radioactive glycosylation labeling require radioactive substrates, which is harmful to biological tissue. Therefore, it is still necessary to develop rapid and simple method for detection of 5-hmC in genomic DNA.(1) In this work, a novel signal-on photoelectrochemical(PEC) immunosensor was fabricated for M.Sss I methyltransfease(MTase) activity analysis and inhibitor screening based on an in situ electron donor producing strategy, where the anti-5-methylcytosine antibody was selected as DNA CpG methylation recognition unit, gold nanoparticle labeled streptavidin(SA-AuNPs) as signal amplification unit and alkaline phosphatase conjugated biotin(ALP-Biotin) as enzymatic unit. In the presence of M.Sss I MTase, hairpin DNA1 containing the palindromic sequences of 5'-CCGG-3' in its stem was methylated. After hybridization with biotin-conjugated DNA2, the stem-loop structure of the hairpin DNA1 was unfolded and the duplex strand DNA(dsDNA) was formed. Then, the dsDNA was captured on the surface of anti-5-methylcytosine antibody modified electrode through the specific immuno-reaction. Afterwards, SA-AuNPs and ALP-Biotin was further captured on the electrode surface through the specific reaction between biotin and streptavidin. Under the catalysis effect of ALP towards ascorbic acid 2-phosphate trisodium salt(AAP), ascorbic acid(AA) was in situ produced as electron donor and a strong PEC response was obtained. The fabricated biosensor showed high detection sensitivity with low detection limit of 0.33 unit/mL for M.SssI MTase. Furthermore, the inhibition research suggested that RG108 could inhibit the M.SssI MTase activity with the IC50 value of 152.54 nM.(2) We describe an electrochemical bioassay for the determination of the activity of methyltransferase(MTase) activity, and for screening this enzyme's inhibitors. It is based on the conjugation of a hemin to a G-quadruplex that enables enzymatic signal amplification with the aid of exonuclease III(Exo III). In the first step, double-stranded DNA containing the quadruplex-forming oligomer is assembled on the surface of a gold electrode and then methylated by DNA adenine methyltransferase(DAM). After cleaved by endonuclease DpnI, the methylated DNA is digested by Exo III and the quadruplex-forming oligomers will be liberated. This leads to the formation of a hemin/G-quadruplex(in presence of hemin and of potassium ions). The hemin/G-quadruplex could catalyze the oxidization of hydroquinone by H2O2 and the benzoquinone was formed to generate electrochemical signal. Finally, the gold electrode modified with reduced graphene oxide was used as working electrode for electrochemical measure. The method has a detection limit of 0.31 unit?mL-1. A study on the inhibition of MTase showed it was inhibited by epicatechin with an IC50 value of 157 ?M.(3) Here, A novel electrochemical immunosensor was fabricated for 5-hydroxymethylcytosine(5-hmC) quantitative detection in genomic DNA of breast cancer tissue based on anti-5-hmC antibody, biotin functionalized phos-tag(Phos-tag-biotin) and avidin functionalized alkaline phosphatase(avidin-ALP), where anti-5-hmC antibody was selected as 5-hmC recognition unit, phos-tag-biotin was used as link unit, and avidin-ALP was used as enzymatic signal amplification unit. First, 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate(5-hm-dCTP) was captured on the surface of anti-5-hmC antibody modified electrode through the specific immuno-reaction. Afterwards, the phos-tag-biotin could be assembled on the electrode surface through the specific recognition effect of phos-tag towards phosphate group. Subsequently, based on the specific reaction between biotin and avidin, avdin-ALP could be further immobilized on the electrode surface successively. Under the catalytic effect of ALP towards p-nitrophenyl phosphate disodium salt, p-nitrophenol was produced as electrochemical activity molecule. The fabricated immunosensor presented high detection sensitivity with low detection limit of 0.032 nM. Furthermore, the electrochemical immunosensor quite successfully demonstrated that the levels of 5-hmC are dramatically reduced in human breast cancer tissue when compared with normal tissue.(4) In this paper, a novel signal-on photoelectrochemical(PEC) biosensor was papered for detecting 5-hydroxymethylcytosine(5-hmC) based on in situ electron donor producing strategy and all wavelengths of light irradiation. Firstly, the amino modified double DNA strand containing 5-hmC was captured on the carboxyl functionalized magnetic bead surface via amide linkage. After treatment with cysteamine in the presence of M.HhaI, the dsDNA with 5-hmC could transformed to amino-derivatized DNA. Subsequently, NHS-biotin could be labeled on the amino-derivative dsDNA and further reacted with avidin conjugated alkaline phosphatase(avidin-ALP). Under the catalysis effect of ALP towards ascorbicacid 2-phosphatetrisodium salt(AAP), ascorbic acid(AA) was in situ produced as electron donor. After magnetic separation, the biosensor can achieved all wavelengths of light irritation and a strong PEC response was obtained using a CdS/ITO as working electrode. The prepared biosensor showed high detection sensitivity with low detection limit of 0.167 nM for 5-hmC. |
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