The Research On Thrombin Aptamers Using Single-molecule Magnetic Tweezers

As thrombin aptamer?HD1?HD1,as one of the"star"aptamers,can control the activity of thrombin and regulating the clotting process is expected to be a biological nontoxic nucleic acid drug,and thus has attracted lots of attention.On the basis of the monomeric thrombin aptamers HD1,repeated long-chain HD1 thrombin aptamers and bivalent thrombin aptamers?HD1 sequences combine with HD22 sequences?are generated,which are expected to further enhance the affinity efficiency and expand the application field.Single-molecule study on the behaviors of these thrombin aptamers will facilitate disclosing the mechanism thrombin binding and provide theoretical basis for the better design of nucleic acid drugs or sensors.Magnetic tweezers?MT?-based single molecule force spectroscopy?SMFS?has super stability in tiny force control and became a very useful technique for the investigation of DNA-protein interactions.In the first chapter,the MT-SMFS was introduced in detail including the structure and principle of magnetic tweezers,the data acquisition and analysis,identification of single-molecule stretching,the force calibration method based on double exponential function.In the following chapters,the MT-SMFS was used as the major characterization method to study advanced structures,mechanical stability,the folding kinetics and the cooperative working mechanism of thrombin aptamers.In the second chapter,using covalent coupling and the force calibration method that is based on double exponential function,a magnetic tweezers based single-molecule manipulation and detection system has been constructed,which laid a foundation for the research in the following chapters.Using 2700 bp double-stranded DNA and?GB1?₈ protein as model systems,the covalent coupling method,force accuracy and spatial resolution of magnetic tweezers were verified.The stability of magnetic tweezers-sample system was improved greatly,as a result the same single-molecule can be manipulated continuously for up to 12 hours.The accuracy of magnetic tweezers system was tested by force fingerprint of double-stranded DNA and?GB1?₈protein.In addition,the force fingerprint was also used to determine the spatial resolution of magnetic tweezers.By studying the folding/unfolding process of?GB1?₈protein,the advantage of magnetic tweezers in minute force control has been proved.In the third chapter,the folding/unfolding processes of monomeric and dimeric thrombin aptamer HD1 were studied,and the rod-like structure of thrombin aptamer was discovered for the first time in HD1 dimer.The dsDNA-ssDNA-dsDNA system was constructed by linking the two segments of DNA handles with HD1 monomer and HD1 dimer respectively.By comparing the unfolding modes of HD1 monomer and HD1 dimer,the"rod-like aptamer"structures of HD1 dimer was identified for the first time.In addition,the MT result showed that thrombin-binding facilitates the formation of rod-like aptamer structures.Furthermore,the study of the relationship between incubation time and refolding probability of HD1 monomer by magnetic tweezers showed that the folding probability of HD1 structure shows a logarithmic dependence on the incubation time:the folding probability exhibit rapid growth in0-30s,slow growth rate after 30s,and the saturation after 60s.In the fourth chapter,the effects of copy number,spacer length and thrombin on the rod-like HD1structures were studied using repeated long-chain HD1 as a model system.We developed the polymerase change-rolling circle amplification?PC-RCA?synthetic method and prepared two-end modified long-chain ssDNA containing multiple repeated HD1 sequences.MT-based SMFS and atomic force microscopy?AFM?imaging have been used to explore the structure of such long-chain ssDNA,and the results confirmed the presence of rod-like HD1 structures obtained in the dimeric HD1.The existence of the rod-like HD1 structures appeared in the repeat sequence may indicate that such rod-like folding structure is one of the causes of genetic diseases,such as Huntington's disease,due to abnormal copy numbers of repeated sequences in the human body.The increase of the spacer length can give the thrombin aptamers more freedom and thus increase the probability for the formation of rod-like structure without affecting their mechanical stability.The presence of thrombin can further promote the formation of rod-like aptamers.In the fifth chapter,the cooperative mechanism of two structural units was studied in bivalent thrombin aptamer(HD1-T₁₅-HD22)as a model system by SMFS based on magnetic tweezers.The dsDNA-ssDNA(HD1-T₁₅-HD22)-dsDNA system was constructed by linking the two segments of DNA handles with bivalent thrombin aptamer.During the experiment,the unfolding and folding signals of bivalent thrombin aptamers were successfully identified by SMFS and the kinetic behavior of HD1 and HD22 components was distinguished by the difference of their complete unfolding length.On this basis,we studied the cooperative and dissociation mechanism of two structural units in the bivalent thrombin aptamer.It provides a direction for the design and optimization of high-performance thrombin aptamers.