| The non-covalent interaction between streptavidin and biotin is really strong which has been applied to various fields such as molecular biology,immunology,biotechnology.The unique binding ability of STV to Biotin is closely related to its structure and its physicochemical properties.People also invented many methods to study the structure and function of STV molecules before.However,most of them are group experiments,and some information of STV molecules may be concealed in the population.Single-molecule technology can be used as a powerful tool to study the complex behavior of biomolecules and to discover individual features that are covered by the effects of group average effects.It is still impossible to study the physicochemical properties of STV molecules at the single molecule level.Compared with other single-molecule technology methods,Atomic Force Microscopy(AFM)has great advantages,for example,the samples don't need to be labeled(such as fluorescence),imaging with high resolution,imaging in liquid environment,etc.,AFM is a main means to study the chemical properties of single biomolecule,such as the structure,function,and dynamic properties.PeakForce Quantitative Nanomechanics Mode is a AFM imaging mode which can measure the mechanical properties of materials in nanoscale while high-resolution imaging.STV molecules is small,soft,and difficult to be operated at surface.It's more challenging to explore its physicochemical properties at the single molecule level.This study used the characteristics of DNA origami addressable,,to study the physical and chemical properties of STV.This study used the characteristics of DNA origami addressability to position Biotin on origami,and selectively fixed the STV at the position set by the origami to study the physicochemical properties of STV.The work mainly includes the following parts:Firstly,a high-resolution imaging method of biomolecule based on DNA origami was designed to achieve high-resolution imaging of a single STV molecule.A single STV has an "hourglass-shaped" structure,and the dimensions of STV in the xyz direction are similar which are between 3 nm to 6 nm.At the same time,this work explored that the morphology of STV molecules changed under different forces.It was found that the best imaging force is 90 pN.As the force increased,the STV molecules were gradually “squashed”,even dissociate from biotin molecules due to deformation;Secondly,this work explored the binding efficiencies of STV at different distances in the site of biotin,different numbers of the sites of Biotin,and different length of the short chain extension of the site.It was found that the bivalent binding of STV occurred relatively close to the biotin distance of 2 nm;the monovalent binding affinity of STV is lower than bivalent.An extension of the modified short chain will increase the freedom of biotin activity,thereby increasing the binding efficiency of STV.This part of work provided experimental basis for modification of molecules on DNA origami and exploration of STV-Biotin system at DNA origami interface;Thirdly,using the DNA origami addressing and positioning function and BiotinSTV molecular specific recognition immobilized the STV molecule at a specific position on the DNA origami plane,and the modulus of the STV molecule was measured at single molecule level,which is about 75 MPa.It can be seen as a reference for the modulus measurement of biomolecules in small sizes.This method based on DNA origami to explore the physicochemical properties of STV is simple and convenient,and provides new ideas for studying the morphology and function of other biomolecules. |
PDF Full Text Request