Novel Method to Detect DNA Methylation Using Gold Nanoparticles Coupled with Enzyme-Linkage Reactions

Abstract

DNA methylation, catalyzed by methylases, plays a critical role in many biological processes, and methylases have been regarded as promising targets for antimicrobial drugs. In this paper, we propose a simple and sensitive colorimetric assay method to detect the activity of methylases so as to monitor DNA methylation using DNA-modified gold nanoparticles (AuNPs) coupled with enzyme-linkage reactions. The duplex DNA molecules modified on the surface of AuNPs are first methylated by DNA adenine methylation (Dam) methyltransferase (MTase) and then cut by methylation-sensitive restriction endonuclease Dpn I. Removal of duplex from the AuNP surfaces by the methylation/cleavage process will destabilize the nanoparticles, resulting in aggregation of AuNPs and a red-to-blue color change. Consequently, the enzyme activity of Dam MTase can be assayed and DNA methylation can be detected. Furthermore, this study may provide a sensitive platform to screen inhibitors for Dam MTase.

Polymerase/nicking enzyme synergetic isothermal quadratic DNA machine and its application for one-step amplified biosensing of lead (II) ions at femtomole level and DNA methyltransferase

“…A low detection limit of 0.05 U ml − 1 was achieved, which is superior to that of most previously reported methods. [36][37][38] In addition, the high selectivity for Dam MTase against other enzymes, including M.SssI MTase and DNA ligase, was successfully demonstrated (Supplementary Figure S6). …”

Magnetic immunoassay using CdSe/ZnS quantum dots as fluorescent probes to detect the level of DNA methyltransferase 1 in human serum sample

“…18 Furthermore, in order to simplify the process, shorten the analysis time, improve the sensitivity, and lower the cost, many new techniques and methods such as fluorescence methods based on hairpin DNA probe, [19][20][21] enzyme immunoassay based on gold nanoparticles, 22,23 surface-enhanced Raman spectroscopy, 24,25 chemiluminescence assay, 26,27 electrochemical methods, [28][29][30][31] and so on have been designed to evaluate the activity of DNA MTase.…”

Gold Nanoparticle-Based Colorimetric Sensors for Detection of DNA and Small Molecules

“…After DNA modification, surface density can be decreased via ligand-exchange reaction by using "backfilling" molecules, thiol molecules that are shorter than the DNA probes. 163,171,173,179,180 Specifically, the gold substrate is exposed to thiolated ssDNA solution first, followed by exposure to a backfilling molecule solution, which form a mixed monolayer of DNA and thiolated molecules. This ligand-exchange process removes both non-specifically adsorbed DNA and some of the covalently attached DNA from the surface, depending on the exposure time 28 in diluent solution or the concentration of backfilling molecules.…”

“…74,161,177,180 For example, in one study, an adenosine-binding aptamer was hybridized with a short complementary AuNP-conjugated DNA, which was stable and well dispersed in the solution. The aptamer strands underwent structure-switching upon binding adenosine, leading to their release from the AuNPs.…”

“…74 Song and Liu reported colorimetric detection of DNA methylation using DNA-AuNPs in a restriction endonuclease-based assay. 180 They treated the DNA-AuNPs with MCH for 8 minutes to decrease the surface density from ∼90…”