The Study Of Solvent Effects On DNA Condensation In Single Molecule Level

DNA condensation is the core of molecular biology.Single molecule manipulation is the main method of the molecule biology. In this paper, we study DNA condensation by sinle molecule magnetic tweezer(sMT) and atomic force microscopy(AFM). The main contents are as fellows:1. We performed systematic studies of lambda-DNA condensation on mica surfaces induced by alcohol and hexammine cobalt (III) [Co(NH₃)₆³⁺] using AFM. The critical condensation concentration for Co(NH₃)₆³⁺ was found to be about 10μM; the DNA molecules extended freely on mica when the concentration was below the critical value. The morphology of condensed DNA became more compact with increasing concentration. At about 500μM Co(NH₃)₆³⁺ concentration, no condensation patterns could be observed due to charge inversion of the compact structures resulting in failure of adhesion to the positively charged surfaces. The critical concentration for alcohol was about 15% (v/v). At this concentration, a few intramolecular loops could be observed in the AFM images. With increasing ethanol concentration the condensation pattern became more complicated ranging from flower-like to pancake-like. When the solution contained both alcohol and hexammine cobalt (III), DNA condensation patterns could be observed even when the concentrations of the two condensation agents were lower than their critical values. We observed this phenomenon by adding mixtures of 10% alcohol and 8μM hexammine cobalt (III) to DNA solutions. The condensation patterns were more compact than those of the condensation agents separately. Typical toroids were found at an appropriate alcohol and hexammine cobalt (III) concentration. The collaborative condensation phenomenon was analyzed by electrostatic interaction and charge neutralization.2. As a widely used precipitation agent for DNA extraction, ethanol is used to induce single molecule DNA condensation. This process is studied with force-measuring MT and AFM. Our experiments provide direct evidence of the metastable intermediate racquet states in DNA collapse induced by ethanol. The measured condensing force is less than 0.2pN even at 50% ethanol concentration, which is much less than those induced by multivalent cations and cationic surfactants. We confirmed the A-B transition of DNA in ethanol and found that the tensile modulus of A-form DNA is larger than that of B-form. Single molecule pulling experiment shows very different features of neutral ethanol from those of multivalent cations. The pulling curve contains a wide range of step sizes, ranging from tens of nanometers to a few micrometers, contrasting with the relatively uniform interval (about 200 nm) in multivalent cations. Meanwhile, the persistence length of DNA decreases monotonically with the increasing ethanol concentration. The condensing morphologies by the weak attraction of DNA segments in the less polar solvent are loose and flowerlike structures composed of many annealed irregular racquets. The analysis of pulling experiments is supported by AFM direct imaging. We concluded that the dominant factor in DNA condensation induced by ethanol is solvent exclusion rather than charge neutralization correlation effect.3. Proteins interacting at multiple sites on DNA via looping play an important role on many fundamental biochemical processes. Restriction endonuclease BspMI that must bind at two recognition sites for efficient activity is a useful model system for studying such interactions. In our experiments, Ca²⁺ was substituted for the normal enzyme cofactor Mg²⁺. Under these conditions it was found that specific binding occurs but cleavage does not. Enzyme in tetramer way interact DNA in the manner of binding one site or forming loops on two sites. AFM images of DNA molecules adsorbed on mica in our experiments for different BspMI concentrations. The present of the enzyme at the specific position can be easily observed. It is worth noting that nonspecific binding to DNA represent less than 8% of the total BspMI-DNA complexes. When the enzyme concentration is increased, the high binding rate and the rate of forming loops were occurred on mica surface. The statistics on loop sizes about BspMI-DNA complexes were reported in this study.