Single-molecule Force Spectroscopy Based On DNA Origami

Force is a signal that cannot be ignored in the process of biomolecular inter-action.It can directly reflect the interaction intensity between two molecules.From cell movement to DNA replication and isolation,from peptide folding to antigen-antibody binding,almost all biological processes are driven by molecular-level forces.However,understanding the interactions between substances and life processes from the single molecule level has the advantages of avoiding the average effect of cluster research,capturing transient intermediates,and characterizing subgroups in heterogeneous systems.At present,the more common single-molecule manipulation technologies mainly include magnetic enthalpy,aperture and single-molecule force spectrum technology(AFM-SMFS)based on atomic force microscopy.Compared with optical and magnetic enthalpy,AFM-SMFS technology combines the advantages of highresolution imaging and mechanical measurement,making it the most effective means of studying intermolecular or intramolecular interactions.However,conventional AFM-SMFS requires separate preparation of each sample and cantilever,increasing the amount of experimental work,resulting in calibration uncertainty and increasing the number of undetermined environmental variables.This makes the subsequent data screening and data analysis of the single-molecule force spectrum large and cumbersome.However,by modification,the two ligands can be modified on the AFM probe and the DNA origami with addressability.The advantage is that the addressable positioning function of DNA origami is more purposeful to detect the interaction between two molecules,making the data more reliable and easier to analyze.The main work of this thesis is to modify the two complementary DNA single strands on the AFM probe and DNA origami,and establish a force spectrum measurement method based on the DNA origami with positioning function.The nano-precision positioning function of DNA origami and the mechanical measurement function based on AFM singlemolecule force spectrum were used to realize high-throughput detection of continuous base mismatch in one experiment and visually quantitative determination of single molecule interactions between streptavidin and biotin.The specific research content as follows: Part 1: Characterization of single molecular structure on DNA origami and its influence of spatial difference on molecular reaction.Using DNA origami as the nanolocalization template of the target molecule Digoxin,the number of molecules and the molecular spacing are precisely controlled,so that the interaction between Digoxin and IgG molecules can be detected from the valence state and the spatial position.Part 2: Inspired by DNA origami to support the precise measurement of biomolecular interactions and structures,a single molecular force spectroscopy method based on DNA origami was developed.Based on the DNA origami,a plurality of modification sites are selected on the DNA origami according to the geometry of the DNA origami,and a short strand of the target DNA is extended at the 5’ end of the oligonucleotide chain of the selected modification site.The short strand of the target DNA can be complementary to the short strand of DNA immobilized on the AFM probe;then the interaction between the two complementary DNA short strands on the DNA origami and the AFM probe is performed by atomic force microscopy single molecular force spectroscopy.The assay was performed and specific force-distance curve data was collected.The method provides an accurate high-throughput single-molecule force spectroscopy method with high detection efficiency,specificity and precision.This simple and efficient high-throughput single-molecule force spectrum method is a quantitative measurement of single-molecule mechanics.Provides an efficient and accurate measurement method.Part 3: The detection of base mismatches was achieved using the created Parallel Single-molecule Force AFM Spectroscopy(P-SMFS).Using programmatic DNA origami,with P-SMFS,we can successfully distinguish between six different target DNAs with no or multiple base mismatches on the origami in a matter of minutes.In particular,different targets with only a single base pair mismatch are clearly distinguished by the difference in the unbinding force of only 4 pN,which is a sensitivity beyond the limits of the conventional AFM-SMFS method.Collectively,our approach demonstrates that programmed DNA origami carrying specific target molecules provides a simple and reliable platform for multiplex measurement of AFMSMFS for high-throughput screening of nanomechanical properties of individual biomolecules.Part 4: Visualization of the single molecule interaction between streptavidin and biotin.Due to the high affinity between streptavidin and biotin and the relatively long bond lifetime.It is often used as a linker in a force spectrum measurement system,so it is necessary to clarify the influence of various factors on the strength of interaction between streptavidin and biotin.In order to be able to visualize quantitative measurements of streptavidin and biotin.We used DNA-backed paper with addressable function as a substrate to immobilize streptavidin molecules,immobilized biotin molecules on AFM probes,and vertically aligned biotinylated AFM probes onto chains on origami.Mold avidin and single molecule force spectrum can quantitatively obtain the strength of interaction between streptavidin and biotin,and the process is visualized.This method can be used for streptavidin and biotin in the future.The system provides a deep understanding of the single-molecule force spectrum of the linker.