Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A sensitive and selective electrochemical method was developed for the detection of DNA methylation, determination of DNA methyltransferase (MTase) activity and screening of MTase inhibitor. Methylene blue (MB) was employed as electrochemical indicator and DNA-modified gold nanoparticles (AuNPs) were used as signal amplification unit because the DNA strands in this composite have strong adsorption ability for MB. First, the thiolated single-stranded DNA S1 was self-assembled on gold electrode, hybridization between the lower portion of DNA S1 and its complementary DNA S2 formed an identical double-stranded tetranucleotide target sequence for both DNA adenine methylation (Dam) MTase and methylation-resistant endonuclease Mbo I, then the upper portion of DNA S1 was hybridized with its complementary DNA S3 modified on AuNPs to bring the DNA S3-AuNPs amplification units onto the electrode. The DNA S1/S2/S3-AuNPs bioconjugate has lots of DNA strands, and they can adsorb abundant MB. Mbo I endounuclease could not cleave the identical target sequence after it was methylated by Dam MTase. On the contrary, the sequence without methylation could be cleaved, which would decrease the amount of adsorbed MB. The presence of redox-active MB was detected electrochemically by differential pulse voltammetry (DPV). Thus, the activity of Dam MTase and methylation status were sensitively converted to the DNA S3-AuNPs amplified DPV signals. The DPV signal demonstrated a linear relationship with logarithm of Dam concentration ranging from 0.075 to 30U/mL, achieving a detection limit of 0.02U/mL (S/N=3). Also, screening of Dam MTase inhibitor 5-fluorouracil was successfully investigated using this fabricated sensor.
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PMID:DNA-AuNPs based signal amplification for highly sensitive detection of DNA methylation, methyltransferase activity and inhibitor screening. 2461 68

In this work, a highly sensitive electrochemical DNA methyltransferase (MTase) activity assay was fabricated with DNA-gold nanoparticles (Au NPs) network as signal amplification unit and an easy assembly method by the linkage of benzenedithiol bridge. By two complementary AuNPs modified single-stranded DNA, DNA-gold nanoparticles network was self-assembled. With the linkage of benzenedithiol bridge, the DNA network structure was immobilized on the surface of gold electrode through the covalent Au-S bond. In the presence of Dam MTase, the special sites of DNA-AuNPs network were methylated and could not be digested by restriction endonuclease Mbo I. Thus the loaded electrochemical indicator Methylene blue (MB) was MB molecules still remained on the DNA-Au NPs network. The electrochemical response depended on the methylated degree, which could be used to detect MTase activity. By the differential pulse voltammetry (DPV), it was demonstrated that a linear relationship between the DPV response and logarithm of Dam concentration ranged from 0.075 to 30 U/mL, achieving a low detection limit of 0.02 U/mL. The use of benzenedithiol avoided the direct incubation of the solid electrode with the capture DNA probe under complex and harsh conditions. Therefore the immobilization of DNA-AuNPs network was easy to be carried out, which is favorable for the specially high stability and reproducibility of the electrochemical biosensor.
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PMID:DNA-gold nanoparticles network based electrochemical biosensors for DNA MTase activity. 2699 15