Construction of a single quantum dot nanosensor with the capability of sensing methylcytosine sites for sensitive quantification of methyltransferase

摘要

CpG island methylation plays an important role in diverse biological processes including the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. Due to the dependence of DNA methylation on DNA methyltransferase (MTase) activity, DNA MTases have become the potential targets in anticancer therapy. Herein we demonstrate for the first time the construction of a single quantum dot (QD) nanosensor with the capability of sensing methylcytosine sites for sensitive quantification of M.SssI CpG methyltransferase (M.SssI MTase). We design a biotin-/phosphate-modified double-stranded DNA (dsDNA) substrate with a 5 '-G-C-G-mC-3 '/3 '-mC-G-mC-G-5 ' site for sensing M.SssI MTase. In the presence of M.SssI MTase, the methylation-responsive sequence of the dsDNA substrate is methylated and cleaved by GlaI endonuclease, producing two dsDNA fragments with a free 3 '-OH terminus. In the presence of terminal deoxynucleotidyl transferase (TdT), multiple Cy5-dATPs can be sequentially added to the free 3 '-OH terminus of dsDNA fragments to obtain biotin-/multiple Cy5-labeled dsDNAs. The resultant biotin-/multiple Cy5-labeled dsDNAs can assemble on the surface of the streptavidin-coated QD to obtain a QD-dsDNA-Cy5 nanostructure in which the fluorescence resonance energy transfer (FRET) from the QD to Cy5 can occur. The emission of Cy5 can be simply quantified by single-molecule detection. By the integration of sensing methylcytosine sites and enzymatic polymerization, the sensitivity of this nanosensor has been significantly enhanced. This nanosensor can detect as low as 2.1 x 10(-7) U mu L-1 M.SssI MTase with good selectivity against other cytosine MTases, and it can be further applied for the screening of MTase inhibitors and complex biological sample analysis, holding great potential in clinical diagnosis and drug discovery.