Evaluating The Effect Of Lidocaine And DNase ? On Biomolecular Interaction Using Atomic Force Microscopy

The characterization of biomolecular interaction is paramount important for basic biomedicine and affinity-based analytics.Generally,most of the research on biomolecular interaction is in near-physiological condition,leaving out of consideration about the influence of external factors.However,the environment in vivo is complex and external factors,which include electric field,temperature,and metal ion,medicine and enzyme,could influence biomolecular interaction.Hence,it is indispensable to investigate the effect of external factors on the interaction of the biomolecule.Evaluating the effect of external factors on the interaction of biomolecule at single molecule level would be helpful to the analysis of binding mechanism and dissociation processes of biomolecular interaction.As a unique tool for monitoring the forces between biomolecules at single molecule level,Atomic force microscopy(AFM)can provide not only new insights into biomolecular recognition process,but also the information on the dissociation dynamics of biomolecule.In this thesis,the effect of lidocaine and DNase ? on the interaction of biomolecule was explored by AFM.More details were as following:1.Evaluating the effect of lidocaine on the interactions of CRP with its aptamer and antibody by atomic force microscopyAptamer or antibody was first covalently attached to the AFM tip through a PEG linker.And CRP was immobilized on the gold substrate by physical absorption.Then,the interactions of CRP with its aptamer and antibody under different temperature were explored before and after CRP-coated gold substrate with lidocaine treatment.The results revealed that lidocaine could reduce the binding probabilities and binding affinities of the CRP-aptamer and the CRP-antibody.An interesting discovery was that lidocaine had differential influences on the dynamic force spectra of the CRP-aptamer and the CRP-antibody.The energy landscape of the CRP-aptamer turned from two activation barriers to one after the treatment of lidocaine,while the one activation barrier in energy landscape of the CRP-antibody almost remained unchanged.In addition,similar results were obtained for 25 ? and 37 ?.In accordance with the result from molecular docking,the reduction of binding probabilities might be due to the binding of lidocaine on CRP.Additionally,the alteration of the dissociation pathway of the CRP-aptamer and the change of binding affinities might be caused by the conformational change of CRP,which was verified through synchronous fluorescence spectroscopy.Furthermore,differential effects of lidocaine on the interactions of CRP-aptamer and CRP-antibody might be attributed to the different dissociation processes and binding sites of the CRP-aptamer and the CRP-antibody and different structures of the aptamer and the antibody.2.Evaluating the effect of DNase ? on the interactions of DNA and MO with its cDNA by atomic force microscopyDNA probe or MO probe of the same sequence were first covalently attached to the AFM tip,and the cDNA was immobilized on the gold substrate.Then,the interactions of MO probe and DNA probe with its cDNA were inverstigated using AFM.The results indicated that both the most probable force and binding probability of MO-cDNA were higher than that of DNA-cDNA.The results derived from dynamic force spectroscopy revealed that there were two activation barriers and one intermediate state in the unbinding process of MO-cDNA and DNA-cDNA complexes.Moreover,the binding capacity of MO and DNA with its cDNA closely related with the activation energy barrier.The effect of ionic strength on the interaction of MO probe and DNA probe with its cDNA was also inverstigated.As the decrease of ionic strength,the most probable force of MO-cDNA and DNA-cDNA decreased.When the ionic strength diluted to a certain critical concentration,the binding probability of MO-cDNA almost remained unchanged,while the binding probability of DNA-cDNA decreased.In addition,the ability of MO and DNA for nucleobase mismatch discrimination was inverstigated.The results revealed that the most probable force of DNA and MO with cDNA containing base mismatch both decreased.The binding probability of the former remained unchanged,but the binding probability of the later reduced with the increase in amount of mismatch base.Moreover,the interaction of MO probe and DNA probe with its cDNA after the functionalized tip with DNase ? treatment was inverstigated using AFM.The most probable force and binding probability of DNA-cDNA were reduced after DNA was treated by DNase ?.However,these paramester of MO-cDNA almost remained unchanged after the treatment of DNase ?.The properties of MO were superior to that of natural nucleic acid in hybrid efficiency,anti-enzymolysis capability and base mismatch discrimination even under low ionic strength,which was attributed to the backbone flexibility and electric neutrality of MO.