Yu.M. Yevdokimov, V.I. Salyanov, M.N. Savvateev, V.A. Dubinskaya, S.G. Skuridin

Double-stranded DNA molecules at phase exclusion from water-salt polyethylene glycol (PEG) containing solutions form cholesteric liquid-crystalline dispersions (CLCDs). DNA CLCD particle can be considered as a «drop» of a concentrated solution of nucleic acid, the structure and properties of which are determined by the value of PEG-solution osmotic pressure. These particles can not be immobilized on a surface of membrane (film), because at low osmotic pressure transition of DNA molecules from condensed (liquid-crystalline) state to isotropic one takes place. This fact dictates a necessity in development of approaches to define the size and shape of DNA CLCD particles. One of them, based on the «cross-linking» of neighboring DNA molecules fixed in the spatial structure of the CLCD particles by artificial nanobridges, results in formation of «rigid» DNA nanoconstructions (NaCs). The formation of «rigid» DNA NaCs opened up a possibility for their immobilization on the surface of the nuclear membrane filter and their study by atomic force microscope. It is also known the other methods of transformation of the «liquid» DNA CLCD particles to «rigid» state. The first method is based on the decrease in solubility of DNA molecules as a result of treatment of CLCD particles with salt solutions of rare earth elements (REE), the second one - on formation of linear clusters in the «free space» between neighboring DNA molecules as a result of treatment of DNA CLCD particles with gold nanoparticles of small size. The goal of the present study is to compare the properties of various «rigid» NaCs based on DNA CLCD particles using atomic force microscopy. The «rigid» NaCs of DNA were prepared using three different methods and immobilized on the nuclear membrane filter (the pore diameter 0.2-0.3 nm). The size and shape of the «rigid» DNA NaCs immobilized on the filter were investigated by scanning atomic force microscopes P47-SPM-MDT and SmartSPM. The obtained data show: 1. the mean size (450-500 nm) of different types of «rigid» DNA NaCs and width of their size distribution are almost the same; 2. "rigid" DNA NaCs have form of spherocylinders despite of the different methods of their preparation; 3. «rigid» DNA NaCs differ in their height; in particular, the height of DNA NaCs, induced by rare earth ions or by formation of nanobridges, markedly differs from the height of the NACs obtained as a result of «cross-linking» of neighboring DNA molecules by Au linear clusters. These results are explained in terms of different stability of the DNA NaC spatial structure in solutions with different osmotic pressure. Indeed, the process of disintegration of the «liquid» ChLCD particles depends on the presence and stability of the «cross-links» between DNA molecules in water-salt solution of a low osmotic pressure. In the water-salt solution with an osmotic pressure below the «critical» value used for washing-out of filter with immobilized the «rigid» DNA NaCs the nanobridges are practically not destroyed. This means that the spatial structure of the «rigid» DNA NaC (and its parameters) is preserved in the same state in which it was created in the initial PEG-containing water-salt solution. A similar situation takes place in the case of «rigid» DNA NaCs based on (DNA-REE) complexes. These structures may also exist in the water-salt solution with an osmotic pressure below the «critical» value. In the case of «rigid» DNA NaCs formed as a result of the diffusion of Au nanoparticles in the «free» space between DNA molecules, forming CLCD particles, and the formation of linear clusters in this space, the situation is different. Such clusters may play a role of «cross-links» between neighboring DNA molecules and stabilize the spatial structure of the «rigid» NAC. Under used experimental conditions used the number of such «cross-links» is small. Under washing-out of the nuclear membrane filter by water-salt solution of low osmotic pressure it is possible to remove loosely connected DNA molecules from the NaC structure. In this case, the height of the «rigid» DNA NaC should decrease, it is observed experimentally. The obtained data show that in some cases the determination of the spatial parameters of DNA nanoconstructions by atomic force microscopy may not be quite correct. This incorrectness is associated with different stability of the spatial structure of «rigid» DNA NaCs in solutions of high and low osmotic pressure, which in turn results in partial disintegration of these nanoobjects.