Carbon nanotube signal amplification for ultrasensitive fluorescence polarization detection of DNA methyltransferase activity and inhibition
| Autor: | Hong Liang, Limin Zhao, Jia Chen, Yong Huang, Shulin Zhao, Zheng-Feng Chen, Kun Hu, Ming Shi |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Site-Specific DNA-Methyltransferase (Adenine-Specific)
Drug Evaluation Preclinical Biomedical Engineering Biophysics Stacking Fluorescence Polarization Biosensing Techniques DNA methyltransferase chemistry.chemical_compound Recognition sequence Electrochemistry Humans Enzyme Inhibitors Enzyme Assays Nanotubes Carbon Hybridization probe General Medicine DNA Methylation Orders of magnitude (mass) Restriction enzyme chemistry Biochemistry DNA Fluorescence anisotropy Biotechnology |
| Zdroj: | Biosensors and Bioelectronics. 54:285-291 |
| ISSN: | 0956-5663 |
| Popis: | A versatile sensing platform based on multiwalled carbon nanotube (MWCNT) signal amplification and fluorescence polarization (FP) is developed for the simple and ultrasensitive monitoring of DNA methyltransferase (MTase) activity and inhibition in homogeneous solution. This method uses a dye-labeled DNA probe that possess a doubled-stranded DNA (dsDNA) part for Mtase and its corresponding restriction endonuclease recognition, and a single-stranded DNA part for binding MWCNTs. In the absence of MTase, the dye-labeled DNA is cleaved by restriction endonuclease, and releases very short DNA carrying the dye that cannot bind to MWCNTs, which has relatively small FP value. However, in the presence of MTase, the specific recognition sequence in the dye-labeled DNA probe is methylated and not cleaved by restriction endonuclease. Thus, the dye-labeled methylated DNA product is adsorbed onto MWCNTs via strong π–π stacking interactions, which leads to a significant increase in the FP value due to the enlargement of the molecular volume of the dye-labeled methylated DNA/MWCNTs complex. This provides the basic of a quantitative measurement of MTase activity. By using the MWCNT signal amplification approach, the detection sensitivity can be significantly improved by two orders of magnitude over the previously reported methods. Moreover, this method also has high specificity and a wide dynamic range of over five orders of magnitude. Additionally, the suitability of this sensing platform for MTase inhibitor screening has also been demonstrated. This approach may serve as a general detection platform for sensitive assay of a variety of DNA MTases and screening potential drugs. |
| Databáze: | OpenAIRE |
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