ABSTRACT
HIGHLIGHTS: DNA kinking is inevitable for the highly anisotropic 1D-1D electrostatic interaction with the one-dimensionally periodically charged surface. The double helical structure of the DNA kinetically trapped on positively charged monomolecular films comprising the lamellar templates is strongly laterally stressed and extremely perturbed at the nanometer scale. The DNA kinetic trapping is not a smooth 3D-> 2D conformational flattening but is a complex nonlinear in-plane mechanical response (bending, tensile and unzipping) driven by the physics beyond the scope of the applicability of the linear worm-like chain approximation. Up to now, the DNA molecule adsorbed on a surface was believed to always preserve its native structure. This belief implies a negligible contribution of lateral surface forces during and after DNA adsorption although their impact has never been elucidated. High-resolution atomic force microscopy was used to observe that stiff DNA molecules kinetically trapped on monomolecular films comprising one-dimensional periodically charged lamellar templates as a single layer or as a sublayer are oversaturated by sharp discontinuous kinks and can also be locally melted and supercoiled. We argue that kink/anti-kink pairs are induced by an overcritical lateral bending stress (> 30 pNnm) inevitable for the highly anisotropic 1D-1D electrostatic interaction of DNA and underlying rows of positive surface charges. In addition, the unexpected kink-inducing mechanical instability in the shape of the template-directed DNA confined between the positively charged lamellar sides is observed indicating the strong impact of helicity. The previously reported anomalously low values of the persistence length of the surface-adsorbed DNA are explained by the impact of the surface-induced low-scale bending. The sites of the local melting and supercoiling are convincingly introduced as other lateral stress-induced structural DNA anomalies by establishing a link with DNA high-force mechanics. The results open up the study in the completely unexplored area of the principally anomalous kinetically trapped DNA surface conformations in which the DNA local mechanical response to the surface-induced spatially modulated lateral electrostatic stress is essentially nonlinear. The underlying rich and complex in-plane nonlinear physics acts at the nanoscale beyond the scope of applicability of the worm-like chain approximation.
ABSTRACT
PURPOSE: To develop a general method for NP fabrication from various proteins with maintenance of biological activity. METHODS: A novel general approach for producing protein nanoparticles (NP) by nanoprecipitation of the protein solutions in 1,1,1,3,3,3-hexafluoroisopropanol is described. Protein NP sizes and shapes were analyzed by dynamic light scattering, scanning electron and atomic force microscopy (SEM and AFM). Chemical composition of the NP was confirmed using ultraviolet (UV) spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and circular dichroism (CD). Biological properties of the NP were analyzed in ELISA, immunofluorescent analysis and lysozyme activity assay. RESULTS: Water-insoluble NP were constructed from globular (bovine serum albumin (BSA), lysozyme, immunoglobulins), fibrillar (fibrinogen) proteins and linear polylysines by means of nanoprecipitation of protein solutions in fluoroalcohols. AFM and SEM revealed NP sizes of 20-250 nm. The NP chemical structure was confirmed by UV spectroscopy, protease digestion and EDX spectroscopy. CD spectra revealed a stable secondary structure of proteins in NP. The UV spectra, microscopy and SDS-PAA gel electrophoresis (PAGE) proved the NP stability at +4°C for 7 months. Co-precipitation of proteins with fluorophores or nanoprecipitation of pre-labeled BSA resulted in fluorescent NP that retained antigenic structures as shown by their binding with specific antibodies. Moreover, NP from monoclonal antibodies could bind with the hepatitis B virus antigen S. Besides that, lysozyme NP could digest bacterial cellular walls. CONCLUSION: Thus, the water-insoluble, stable protein NP were produced by nanoprecipitation without cross-linking and retained ligand-binding and enzymatic activities.
