The Study Of Interactions Between Protein, Polyethyleneimine And DNA Using AFM-based Single Molecule Force Spectroscopy

Single-molecule studies have attracted more and more attentions during recentyears. One reason is that such studies focus on the behavior of specific singlemolecule, which is different from that of traditional ensemble measurements.Another reason is that the hidden details of the dynamics or rare events can becaptured through such studies. With the development of nanotechnology, manytechniques which can be used to observe and manipulate single molecules have beeninvented. Among those single-molecule manipulation techniques, such as glassmicroneedles, biomembrane force probe, magnetic beads, optical tweezers andAFM-based single-molecule spectroscopy (SMFS), AFM-based SMFS has beenused widely in the field of single-molecule study due to its friendly user interface(i.e., easy to use and can work in both aqueous solution and organic solvents).In chapter1, we discussed recent advancements in AFM-based single-moleculeforce spectroscopy field. First, we introduced the principle of the AFM-based SMFSexperiment. Then, we introduced the application of AFM-based SMFS in biologicalsystem. Typical force fingerprints of nucleic acid, protein and polysaccharides werediscussed. Then the application of AFM-based SMFS in investigation of nucleicacid-protein interactions and protein-protein interactions were introduced. Suchinformation on interactions of biological systems is useful in revealing themechanism of many important biological processes.In chapter2, we constructed three recombinant Bacillus subtilis ssb gene andpurified these three recombinant proteins. Then native polyacrylamide gelelectrophoresis were used to investigate the impact of His Tag and cysteine on thestructure of protein and the interaction between protein and DNA. It is found that,His Tag induces the formation of aggregates among recombinant proteins throughsulfhydryl groups in cysteines. However, it doesn't affect the interaction between protein and DNA.In chapter3, interactions between Bacillus subtilis single-stranded DNAbinding proteins (BsSSB) and single-stranded DNA (ssDNA) were systematicallystudied. The effect of different molar ratios between BsSSB and ssDNA on theirbinding modes was first investigated by Electrophoretic Mobility Shift Assays(EMSA). It is found that high molar ratio of BsSSB to ssDNA can produceBsSSB-ssDNA complexes formed in the mode of two proteins binding one65-nt(nucleotide) ssDNA, while low molar ratio facilitates the formation ofBsSSB-ssDNA complexes in the mode of one protein binding one65-nt ssDNA.Furthermore, two binding modes are in dynamic equilibrium. The unbinding force ofBsSSB-ssDNA complexes was measured quantitatively in solutions with differentsalt concentrations by using AFM-based single molecule force spectroscopy (SMFS).Our results show that the unbinding force is about10pN higher at high saltconcentration (0.5M NaCl) than at low salt concentration (0.1M NaCl) and thelifetime of BsSSB-ssDNA complexes at high salt concentration is twice as long asthat of at low salt concentration. These results indicate that more tightly-packedBsSSB-ssDNA complexes can form at high salt (0.5M NaCl) concentration. Inaddition, the results of EMSA show that ssDNA, which has bound to the BsSSB, candissociate from BsSSB in the presence of complementary DNA strand indicating thedynamic nature of BsSSB-ssDNA interactions.In chapter4, interactions between Polyethylenimine (PEI) and double-strandedDNA (dsDNA) were systematically studied by using AFM-based single-moleculeforce spectroscopy. It was illustrated that PEI (branched,25kD) and dsDNA formedmore compact complexes with the increase of PEI concentration. Furthermore theinteractions between PEI and dsDNA were found to be insensitive to the pH change(from pH7.4to pH5.0), although PEI has the Proton Sponge Effect.