Atomic force microscopy of DNA, nucleoproteins and cellular complexes: Theuse of functionalized substrates

Progress towards rapid and simple characterization of biomolecular samples by scanning probe microscopy is impeded mainly by limitations of the curren t approach to sample preparation. We are working on approaches based on che mical functionalization of mica. Treatment of mica with aminopropyltriethox y silane (APTES) makes the surface positively charged (AP-mica) and able to hold DNA in place for imaging, even in water. We have shown that AP-mica i s an appropriate substrate for numerous nucleoprotein complexes as well. Th e AFM images of the complex of DNA with RecA protein are stable and indicat e a structural periodicity for this filament. AP-mica holds strongly such l arge DNA complexes as kinetoplast DNA (kDNA) and is an appropriate substrat e for their imaging with AFM. We have further develop this approach for mak ing hydrophobic substrates. Silylation of mica surface with hexamethyldisil azane (Me-mica) allowed us to get AFM images of chlorosomes, an antenna com plex isolated from green photosynthetic bacteria. Me-mica may be converted into a positively charged substrate after treatment with water solutions of tetraethylammonium bromide or cetyltrimethylammonium bromide. These activa ted surfaces show high activity towards binding the DNA molecules.