The Investigation Of Nucleic Acid-Protein Interactions Using AFM-based Single Molecule Force Spectroscopy

Nucleic acids and proteins are two important biomacromolecules that compose the life, and the interactions between them are the base of many biological phenomena, such as growth, propagation, inheritance, metabolism, and so on. The nano-mechanical detection of nucleic acid-protein interactions 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 microscopy (AFM)-based single molecule force spectroscopy (SMFS) is a very effective technique for the investigation of inter- or intramolecular interactions.In chapter 1, the basic principle of SMFS is introduced in detail, including the analysis of a force curve, the criteria for single chain stretching and dynamic force spectroscopy (DFS). Some recent progresses in SMFS study of biological systems have also been summarized. In the later chapters, we investigated two nucleic acid and protein systems from relatively simple to complex one by using SMFS. These two systems are long double stranded DNA (dsDNA) and single-stranded DNA (ssDNA) binding protein (SSB), together with genetic RNA of tobacco mosaic virus (TMV) and its coat proteins, respectively.In chapter 2, we have revealed the mechanism of force-induced conformation transition of dsDNA and the interactions between (single stranded DNA) ssDNA and SSB proteins. Taking advantage of the character that SSB proteins interact with ssDNA specifically but not with dsDNA, we used a long dsDNA as a probe to investigate the nature of force-induced conformation transition of dsDNA. Our results indicate that dsDNA is partially melted into ssDNA during the overstretching transition (i.e. dsDNA exist as a mixture of the dsDNA and molten ssDNA) at the mechanical force of about 65 pN, and the SSB proteins are able to capture the transient ssDNA fragments slowing down the rehybridization process, causing the hysteresis between stretching and relaxation traces. After relaxation, the SSB proteins can be removed from the ssDNA fragments, and the dsDNA recovers its B-form conformation. We have also systematically investigated the effects of stretching length, waiting time, and salt concentration on the conformation transition of dsDNA and SSB-ssDNA interactions, respectively. The study indicates that electrostatic interaction, intercalating interaction and hydrophobic interaction are the main driving forces for the formation of SSB-ssDNA complexes.In chapter 3, we took a more complicated system, an intact tobacco mosaic virus (TMV), as a model to reveal the interactions between genetic RNA and coat proteins. For the first time, by using an AFM tip, we have pulled the genetic RNA step by step out of the helical groove formed by its protein coat, producing a sawtooth-like plateau, from which the quantitative unbinding force between the RNA and coat proteins is obtained. We have proved that the coat proteins stayed in the protein coat but not being pulled out together with RNA. When the external force is released, the detached RNA fragment can find its way back to the helical protein coat with the help of intact RNA-protein complexes located in the deeper part (i.e., away from the 5'end) of the TMV particle. We have also studied the effects of other factors such as EDTA concentration, pH value and loading rate on the interactions between RNA and coat proteins. The present study extends the force spectroscopy technique to the study of nucleic acid-protein interactions in more complicated biological systems. And the method established here may open a new door toward investigations of the mechanism of virus infection.In chapter 4, we still took tobacco mosaic virus as a model system to investigate the dynamic process of RNA-coat protein interactions in more detail. We explored the dependence of unbinding forces of RNA-coat protein complexes on the loading rate, that is, the rate at which the force is applied to the binding. The experimental data can be fitted well by the Bell-Evans model, which enables us to determine the energy barrier width (xp), off-rate constant (koff), binding lifetime (Ï„) and to describe the energy landscape of RNA-coat protein interactions. The results show that at pH 4.7, the unbinding process is only dominated by one energy barrier stabilizing RNA-coat protein complexes, while at pH 7.0, the unbinding process is dominated by two energy barrier, the extra energy landscape comes from the disordered loop of polypeptide in the wall of the inner channel of TMV.