DNA Methylation And Tumor Cells Were Detected Using DNA Self-assembly Techniques

To develop methods for early diagnosis and effective treatment of cancer has been the goal of scientific research workers. In this study, we created two kinds of self-assembled structures using DNA self-assembly technique. One is a end-to-end(ETE)Au nanorod assembly. We have demonstrated a SPR platform for sensitive DNA methylation detection and Dam MTase activity assay based on cycle amplification strategy of polymerization and nicking reactions and of SPR signal amplification with ETE AuNR assemblies. The other one is a functionalized DNA nanopore. The DNA nanopore can recognize tumor cell effectively and span the cell membrane to kill the target cells. The experimental content could be summarized as follows:1. An Au nanorod(AuNR) assembly-enhanced surface plasmon resonance( SPR)system coupling with polymerization and nicking reactions was developed for amplified detection of DNA methylation and adenine methylation(Dam) methyltransferase(MTase)activity assay.The palindromic duplex DNA probes were firstly methylated by DNA Dam MTase and then cut by methylation-sensitive restriction endonuclease Dpn I. The released sequence worked as primer to induce polymerization and nicking reactions, producing a large amount of linker sequences. Subsequently, end-to-end(ETE) AuNR assemblies are linearly and periodically assembled to the Au chip through the specific interactions between capture probes and linker sequences. The enhancement of the SPR signal was achieved by increasing the refractive index of the surface, due to the high molecular weight of AuNR assemblies.The biosensor showed a good linear relationship between the SPR angle shift and the Dam MTase concentration over a range from 0.5 to 120 U/m L, with a detection limit of 0.2 U/mL. This study provided a sensitive platform to screen inhibitors for Dam MTase and had a great potential to be further applied in early clinical diagnosis.2. A DNA nanopore functionalized with Ramos aptamper and cell-penetrating peptide(TAT) is designed to recognize tumor cells and kill them in a shorter time.The nanopore was composed of a bundle of six DNA duplexes folded from six DNA strands, which areinterconnected by crossovers to add structural stability. For fluorescence imaging, two FAM fluorophores were incorporated into the DNA nanopore terminu through two strands at the5'-positions. On the other terminu of the pore, biotin molecules were modified to interact with biotin-modified Ramos cell aptamer and TAT, the sequence from the HIV virus, through the interaction with streptavidin. The dually conjugated molecule was found to show both improved cellular uptake and efective Romas cell targeting. Fluorescence imaging was performed by confocal laser scanning microscopy(CLSM).The DNA nanopore can be further modified to acquire more useful function. DNA nanostructures can be used for cell biological applications, such as gene transfection, drug permeabilization or targeted killing of diseased cells. This study had a great potential to be further applied in effective treatment of cancer.