Investigating The Binding Force Of Thrombin And Its Aptamer Based On Ultra-Low-Field Optical Atomic Magnetometer

Nucleic acids and proteins, as the most two important biomacromolecules in the life system, play a very important role among various kinds of physiological and metabolic activity. Nucleic acid is the main carrier of genetic information, and protein is the important element in organisms. All the life processes in organism work based on the contact of these biomolecules or interaction with other molecules, which caused the physical and chemical changes. The signal transmission, identification of foreign body, DNA replication, transcription and translation were inseparable from the biomacromolecules interaction. Investigating the interaction between them can contributed to explain the physiological phenomenon, disease diagnosis and drug treatment. It is still a hot and difficult problem now to obtain the precise interaction force between nucleic acids and proteins in the field of current research in biochemistry. Although single molecular force spectrum technique has made great progress in field of biomolecules interactions, the force spectrum resolution remains to be further improved. How to measure the interaction force efficiently between them in different aspects still remains challenging.This paper made a review on this emerging field of interaction force between biological molecules based on different force spectrum technique. With thrombin and its corresponding aptamer(apt15) as the research object, we measure the interaction force between them by direct method and indirect method where the thrombin was free or fixed using a new kind of low field force spectrum technology, which use magnetic particle as molecular probes. Analyze the bonding force and patter of thrombin/apt15 comprehensively through different experiment design.The main researches are as follows:In chapter 1, research progress on aptamer and thrombin interaction were summarized. A series of single molecule manipulation techniques,such as AFM?optical tweezers and magnetic tweezers for studying the interaction of nucleic acid/protein and their latest research progress in application scope are mainly introduced. Finally, we introduced the new development of a new kind of ultra-low field biological force spectrum technology-optical atomic magnetometer and briefly reviews the important applications of this method in the aspects of double strand DNA dissociation, cell imaging, drug screening and so on.In chapter 2, Based on the ultra low field biological force spectroscopy, the interaction between nucleic acid and thrombin in the free state was studied. By designing a series of complementary DNA molecules with different sequence lengths, we can control the formation of double helix structure of aptamer and the complementary DNA and regulate the interaction between the ligands and the thrombin. The change of the bonding force of double stranded DNA with different sequence structure in the presence of thrombin protein was study. The double stranded DNA force spectra was obtained by the ultra low field force spectroscopy techniques. The perturbative force was produced though a high precision centrifugal device and calculated indirectly the bonding force of aptamer and thrombin in free to be about 69 ±4p N.In chapter 3, the bonding force of aptamer and thrombin protein in fixed state was studied. The protein was immobilized on the surface of the substrate without the introduction of complementary DNA, and the binding force of thrombin and aptamer was measured directly to be 80 pN. The kinetic information of dissociation is obtained by changing the incubation time and duration of applied force. The molecular mechanisms of aptamer and thrombin protein and their effects on the dissociation process were discussed. At the same time, the research results are compared with the results of the above experiment.In chapter 4, the effects of different lengths of complementary DNA sequences on the interaction of aptamer and thrombin in fixed state was explored. Through the mechanism of molecular regulation, the interaction between the thrombin and aptamer is changed, and the competitive exchange reaction between the complementary DNA, aptamer and the thrombin was regulated. By measuring the dissociation force of the double stranded DNA, the interaction force between the l thrombin and aptamer was obtained.The results were compared with the previous two methods. It was found that the interaction force of fixed thrombin and aptamer was stronger than that of thr ombin in free state.