Construction Of A Quantum Dot-based Nanosensor For Ultrasensitive Detection Of DNA Methyltransferase

In higher order eukaryotes,the most common DNA modification is the methylation of carbon 5?C5?position of cytosines,predominately in CpG dinucleotides.The CpG island methylation is an important epigenetic mark and plays a critical role in development and differentiation,genome stability,genomic imprinting,and X-chromosome inactivation.Aberrant methylation of CpG islands has been linked to various diseases including cancers.The occurrence of DNA methylation depends mainly on DNA methyltransferase?DNMT?activity.The methods for CpG MTase assay include fluorescent assay,single molecule detection,surface enhanced Raman scattering,surface plasmon resonance,electrochemical immunoassay and electro-chemiluminescence measurement.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.We design a biotin-/phosphate-modified double-stranded DNA?dsDNA?substrate with a5'-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 dsDNA substrate is methylated and cleaved by GlaI endonuclease,producing two dsDNA fragments with 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 the 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.The integration of single-molecule detection with QDs endows this nanosensor with high sensitivity.This nanosensor can detect as low as 2.1×10^-7 U/?L 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 clinic diagnosis and drug discovery.