Visualization Of The Recycled Rolling Circle Polymerization In A Cycling Reaction System With Total Internal Reflection Fluorescence Microscopy

In this study, a DNA-related reaction network, in which rolling circlepolymerization was recycled and repeated, was established to enhance the sensitivityof lysozyme detection, and the operation of this system was visualized using totalinternal reflection fluorescence microscopy (TIRFM): the rolling-circle amplification(RCA), as the fundamental amplification, was repeated due to the target-displacementpolymerization (TDP), resulting in further amplification; the RCA repetition numberwas then multiplied by nicking-polymerization cycle, bringing forth extraamplification.Chapter1introduced the development backgroud in the relative field. It mainlyelaborated on single-molecule research progress at home and abroad, principles of totalinternal reflection fluorescence microscopy(TIRF) and rolling circleamplification(RCA). Applications of TIRF and RCA were reviewed. It also illustratedon lysozyme and DNA molecular machine. Thus main subject of this thesis wasintroduced: Visualization of the recycled rolling circle polymerization in a cyclingreaction system with Total Internal Reflection Fluorescence Microscopy.In chapter2, a multiplex amplification system with three amplification layers wasestablished to enhance the sensitivity of detection of lysozyme. The amplificationconsisted of three amplification layers: the fundamental amplification was provided byrolling-circle amplification; the further amplification was provided bytarget-displacement polymerization that made RCA to operate in the cycle mode; and extra amplification was provided by nicking-polymerization cycle. The rolling-circleamplification was cycled by target-displacement polymerization and furtherexponentially amplified by the feed-back from nicking-polymerization cycle. All thereactions originated from a single stimulus: the aptameric recognition of lysozyme;and every layer of reaction resulted in the RCA.Due to the multi-layer amplification was all superposed on the RCA, which itselfwas a prominent amplification, the whole amplification system exhibited a ratherexcellent amplification effect: a detection limit of10-15M grade for lysozyme wasobtained with three DNA reaction cycles, and a specialized selectivity that only thedesired target, lysozyme, could produce prominent signal that differ from the blankresponse.In chapter3, RCA was chosen as the model process to be studied by directvisualization approach. The RCA was designed as a cycling reaction system, takingadvantage of our ongoing study about the reaction network of cycling DNA reactions:RCA was designed as a cycled reaction systerm., combining RCA with other reportedDNA-related processes was attempted to further improve the amplification efficiency.Involved in this work was target-displacement polymerization (TDP), in which a target,which is not nucleic acid, was bound to its aptamer, and then be displaced during DNApolymerization using the aptamer sequence as the template; there was also“polymerization/nicking cycle” developed by Itamar Willner’s group, in whichdouble-stranded DNA (dsDNA) can be nicked at a specific recognition site by arelevant nicking endonuclease (NEase), and the nicking site can then act as a startingpoint for a strand-replacement polymerization, causing the repetitvely releasing of asingle-stranded DNA (ssDNA) and the recovering of the dsDNA.Since the RCA yields a long DNA strand which would be micrometers in length,it would be an idealistic object for the TIRFM study.