Application Of DNA Molecular Machine In The Amplified Dection Of Tumor Markers

MicroRNAs (miRNAs), a class of endogenously non-protein coding RNA molecules about18-24nucleotide long, that can be found in plant, human, and animal tissues play key roles in post-transcriptional gene regulation through binding to the3’ UTR of mRNA targets, resulting in mRNA degradation or repression. Considering the great significance of microRNAs (miRNAs) in cancer detection and typing, the development of sensitive, specific, quantitative, and low-cost methods for the assay of expression levels of miRNAs is desirable. Molecular beacon (MB), specifically designed DNA hairpin structure, has been widely applied in biosensor development, achieving the detection of a broad range of targets from DNA, RNA, protein, metal ion to even tumor cells.This thesis can be summarized as the following two parts.1、Dumbbell Probe-Mediated Cascade Isothermal Amplification for Label-Free Detection of Zeptomole microRNA and Its Application to Real Sample AssayWe describe a highly efficient amplification platform for ultrasensitive analysis of miRNA (taking let-7a miRNA as a model analyte) based on a dumbbell probe-mediated cascade isothermal amplification (DP-CIA) strategy. The method relies on the circularization of dumbbell probe by binding target miRNA, followed by rolling circle amplification (RCA) reaction and an autonomous DNA machine performed by nicking/polymerization/displacement cycles that continuously produces single-stranded G-quadruplex to assemble with hemin to generate a color signal. In terms of the high sensitivity (as low as1zmol), wide dynamic range (covering9orders of magnitude), good specificity (even single-base difference) and easy operation (one probe and three enzymes), the proposed label-free assay is successfully applied to direct detection of let-7a miRNA in real sample (total RNA extracted from human lung tissue), demonstrating an attractive alternative for miRNA analysis for gene expression profiling and molecular diagnostics, particularly for early cancer diagnosis.2、The studies of label-free detection of target DNA、Ramos cell、lysozyme based on the self-assembly of DNA molecular machine.We describe a highly efficient amplification platform for ultrasensitive analysis of target DNA based on DNA molecular machines amplification strategy.The hairpin structure of beacon1is opened by binding target DNA, then hybridized with beacon2、circle1, The circularization of circle1is performed by adding T4DNA ligase. followed by rolling circle amplification (RCA) reaction and an autonomous DNA machine performed by nicking-polymerization cycles that continuously produces single-stranded G-quadruplex to assemble with hemin to generate a color signal with ABTS2--H2O2.The proposed assay is successfully applied to direct detection of Ramos cell、lysozyme.