Investigation Of The Interactions Between Aie Molecule,protein And Nucleic Acid Using Afm-based Single Molecule Force Spectroscopy

Nucleic acids are important biomacromolecules in living organisms, and they are one of the most basic substances in life. They are not only the basic genetic material, but also occupies an important position in the protein synthesis, and play a decisive role in the growth, inheritance, propagation, variation, and so on. According to the chemical composition they can be divided into two categories: DNA and RNA. The nano- mechanical detection the interactions of nucleic acids and other molecules at single- molecule level will deepen our understanding and eventually gain controls on these biological processes. This is also the key to explore the mysteries of life.Atomic force microscope(AFM)-based single molecule force spectroscopy(SMFS), with a force sensitivity of p N order of magnitude and a spatial resolution below 1 nm, allows the measurement of inter- and intramolecular interactions at single molecule level which is different from the traditional average ensemble assays. And it has been widely in a variety of fields, such as chemistry, biology and physics. In this dissertation, the basic principle of the AFM-based SMFS experiment, including the data collection and analysis, the empirical criteria for single-chain stretching and some recent progresses in SMFS study of bio logical systems were introduced in detail. By using SMFS, we investigated the interactions of double-stranded DNA(ds DNA) with aggregation induced emission(AIE) molecules, together with genetic RNA of tobacco mosaic virus(TMV) and its coat proteins, respectively.In chapter 2, we demonstrated a new method for selective modification of the apex of an AFM probe by using force spectroscopy and interface reaction. First, excess amount of terminal NHS-ester were covalently anchored to the AFM tip surface to form NHS-terminated self-assembled monolayers(SAMs). The NH2-terminated SAMs of 12-aminododecanic acid were formed on the silver coated Si-wafer by soaking them into the methanol solution of 12-aminododecanic acid(H2N(CH2)11COOH). The NHS- modified AFM tip was then brought to interact with the NH2-terminated SAMs on silver surface and the contact force and time were strictly controlled by AFM feed back system. And 12-aminododecanic acid was been picked out from the NH2-terminated SAMs, carboxylic acid groups would then be selectivity attached to the apex of the AFM tip. The reproducibility of this method was confirmed by stretching of long DNA fragment and measuring the contour length of DNA.In chapter 3,we studied the binding modes of double-stranded DN A(ds DNA) with two types of synthetic AIE-active molecules, tetraphenylethene-derived dicationic compounds(cis-TPEDPy, trans-TPEDPy) and anthracene-derived dicationic compounds(DSAI, DSABr-C6), by using single molecule force spectroscopy(SMFS). The results demonstrated that DSAI could intercalate into DNA base pairs strongly, while DSABr-C6 was unable to intercalate into DNA due to the steric hindrance of the alkyl side chains. C is-TPEDPy and trans-TPEDPy could also intercalate into DNA base pairs, but the binding shows strong ionic strength dependence. Multiple binding modes of TPEDPy with ds DN A had been discussed. And the electrostatic interaction enhanced intercalation of cis-TPEDPy with ds DNA had also been revealed.In chapter 4, Using in vitro transcription, origin-of-assembly-sequence(OAS)-containing poly A RN A was prepared and assembled with TMV coat protein(CP) to produce the TMV mutant. We then studied the interactions between poly A RNA and TMV coat protein in the mutated TMV particle, which further confirmed that the interaction of RNA-CP was sequence dependence. Besides, we investigated the influence of tannic acid(a plant virus inhibitor) on TMV disassembly. The experimental data showed that the binding strength of RNA-CP was enhanced greatly with the addition of tannic acid. In addition, the average disassembly/unbinding forces increased linearly with the extension length. These results disclosed the possible mechanism of tannic acid as an antivirus agent: preventing the disassembly of TMV RNA from its protein coat.