Electrogenerated chemiluminescence biosensing method for the detection of DNA methylation and assay of the methyltransferase activity.
A novel electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive detection of DNA methylation and assay of the CpG methyltransferase (M. SssI) activity was developed on basis of enzyme-linkage reactions and ruthenium complex served as an ECL tag. The ECL biosensing electrode was fabricated by self-assembling 5'-thiol modified 32-mer single-strand DNA (ss-DNA)-tagged with ruthenium bis (2,2'-bipyridine) (2,2'-bipyridine-4,4'-dicarboxylic acid)-ethylenediamine on the surface of a gold electrode, and then hybridized with complementary ss-DNA to form duplex DNA (ds-DNA). When M. SssI and S-adenosylmethionine were introduced, all cytosine residues within 5'-CG-3' of ds-DNA on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by HpaII endonuclease, the un-methylated cytosines were cleaved, thus led to decrease ECL signal. The ECL intensity of ECL biosensing electrode is related to the methylation level and M. SssI activity in a fixed concentration HpaII endonuclease. The increased ECL intensity was direct proportion to M. SssI activity in the range from 0.05 to 100 U/mL with a detection limit of 0.02 U/mL. This work demonstrates that the combination of the enzyme-linkage reactions with a highly sensitive ECL technique is a great promising approach for the detection of DNA methylation level, assay of the activity of MTase, and evaluation of the capability of inhibitors for the methyltransferase.
Institute of Analytical Science, Northwest University, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Xi'an, Shaanxi 710069, China. yanli@nwu.edu.cn
Yan
Y
Li
Cancan
C
Huang
Jianbin
J
Zheng
Honglan
H
Qi
D015082:2,2'-Dipyridyl / Q000737:chemistry; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D004566:Electrodes; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D008163:Luminescent Measurements / Q000379:methods*; D012428:Ruthenium / Q000737:chemistry; D012680:Sensitivity and Specificity
D015082:2,2'-Dipyridyl; D012428:Ruthenium; D004247:DNA; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases