Application Of DNA Molecular Machine In The Amplified Dection Of Nucleic Acids

Optical analysis method has become a popular analytical technique owing toits high sensitivity, high selectivity, simplicity, practicality, easy operation, quickand efficient analysis. In this paper, two methods for amplified detection of nucleicacids are proposed based on DNA molecular machine-cross rolling circleamplification based on DNA rotaxane and exonuclease-assisted cascaded recyclingamplification, achieving ultrasensitive and highly selective detection of mRNAextracted from cancerous cells and label-free detection of DNA throughcolorimetric analysis, respectively. Moreover, the feasibility of the two methods isevaluated in complex samples and human serum samples.This thesis can be summarized as the following two parts.1. The studies of mRNA detection extracted from cancerous cells through crossrolling circle amplification based on DNA rotaxaneAn ultrasensitive and highly selective new method for PCR-free mRNAexpression profiling is proposed through a novel cross RCA (C-RCA) processbased on DNA-rotaxane nanostructures. The interlocking approach is used tosynthesize two DNA pseudorotaxane (DPR-I and DPR-II) species as prerequisitesfor the assembly of DNA-rotaxanes (DR). Upon the introduction of target cDNAthat specific for β-actin (ACTB) mRNA, the gap-ring of DPR-I is used as the circletemplate for RCA reaction, which further achieves C-RCA between DPR-I andDPR-II through the replacement of corresponding release-DNA in the presence ofbiotinylated primers and DNA polymerase/dNTPs for the synthesis ofhemin/G-quadruplex HRP-mimicking DNAzyme chains to catalyze the oxidationof2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)(ABTS2-), by H2O2to theABTS·-. Considering the high background induced by hemin itself in ABTS2-/H2O2 UV-vis system,streptavidin-modified magnetic nanoparticles (MNPs) are employedto capture the rolling circle products (RCPs) via biotin-streptavidin specificrecognition, achieving a superlow limit of detection of0.1zmol cDNA which canbe comparable with that of PCR. The proposed assay that is further applied to thedetection of ACTB in mammary cancer cells (MCF-7) may facilitate clinicalapplication.2. The studies of label-free detection of DNA through exonuclease-assistedcascaded recycling amplificationThe network consisting of three kinds of unlabeled stem-loop DNAmolecular beacons (MBs) is activated by target DNA in the presence ofexonuclease-III (Exo-III) that can catalyze the stepwise removal ofmononucleotides from3′-OH termini of double-strand, achievingexonuclease-assisted cascaded recycling amplification (Exo-CRA) for label-freedetection of DNA. Through discussing the optimum reaction conditions (reactiontemperature, the amount of Exo-III, reaction time and so on), the assay achieves adetection limit as low as0.1pM with a wide dynamic range of8orders ofmagnitude. In comparison with the methods that only employs MB1to participateexonuclease-amplification and unamplified process, the sensitivity is improved by~2and~4orders of magnitude, respectively. The assay can well discriminateone-base difference in DNA sequences and has an excellent performance inhuman serum samples.