Research On Electrochemical Biosensing Method For Detection Of DNA Methyltransferase Activity And Inhibitor Screening

Objective:DNA methylation is the most well-known epigenetic modification. It is a process carried out by DNA methyltransferase (MTase), which catalyzes methyl group transfer from the donor S-adenosyl-L-methionine (SAM) to the target adenine or cytosine and produces S-adenosyl-L-homocysteine (AdoHcy) and methylated DNA. Methyltransferase plays an important role in DNA methylation process, the quantity and activity of MTase influences DNA methylation level, and the overexpression of DNA MTase in many cancer cells has been confirmed. The pharmacological inhibitors of DNA MTase have promising applications for antibiotics and anticancer therapeutics. In this research, we took advantage of the excellent properties of nanomaterials to construct an electrochemical biosensor, it can be used for sensitive detection of DNA methylation, methyltransferase activity and inhibitor screening.Methods:1. Preparation of DNA S3-AuNPs bioconjugates:AuNPs were prepared according to a standard citrate method. DNA S3 and AuNPs combined through Au-S bond. The TEM and UV-vis spectrophotometer were used to characterize the morphology of AuNPs and DNA S3-AuNPs.2. Construction of electrochemical biosensor:DNA S1, MCH, BSA, DNA S2 and DNA S3-AuNPs were successively modified on pretreated Au electrode, and each step was characterized by electrochemical methods.3. Optimization of experimental conditions:we investigated the concentration of methylation donor SAM and the concentration of Mbo Ⅰ endonuclease.4. Evaluation of the electrochemical biosensor:such as feasibility, selectivity, linear range, detection limit, the inhibition effect of 5-fluorouracil, precision, repeatability and stability.Results:1. The AuNPs and DNA S3-AuNPs were characterized by TEM and UV-vis spectrophotometer. The TEM image of AuNPs indicated that the AuNPs obtained were spherical and well-distributed. However, the TEM image of DNA S3 modified AuNPs was almost the same as the image of AuNPs. In UV-vis absorption spectra, pure AuNPs showed a characteristic adsorption peak at 519 nm, after being modified with thiolated DNA S3, the characteristic peak of AuNPs underwent a 5 nm red shift to 524 nm. These results demonstrated the successful assembly of DNA S3 onto the AuNPs surface.2. CV and EIS were performed for each step of the working electrode preparation. All the data indicated the successful fabrication of the electrochemical biosensor.3. Optimization of experimental conditions.80 μM of SAM was employed for the sensing system in the following experiments.50 U/mL of Mbo I was chosen for the following experiments.4. The electrochemical biosensor can be used for sensitive and selective detection of DNA methylation, assay of DNA MTase activity, and screening of inhibitors. The linear range was from 0.075 to 30 U/m L and the detection limit was 0.02 U/mL. It also exhibited good precision, repeatability and stability.Conclusions:In conclusion, a sensitive and selective electrochemical biosensor for the detection of DNA methylation, assay of DNA MTase activity, and screening of inhibitors was successfully developed. A wider linear range from 0.075 to 30 U/mL and a relatively lower detection limit of 0.02 U/mL were achieved. Furthermore, the influence of the inhibitor 5-fluorouracil on the activity of Dam MTase was directly measured. In addition, the proposed analysis mode had improved generality and could be extended to the detection of other DNA MTase and the corresponding inhibitor screening by exchanging the corresponding DNA sequence, which had significant value to disease diagnosis and drug development associated with DNA methylation.