▪ Abstract The atomic force microscope operates on surfaces. Since surfaces occupy much of the space in living organisms, surface biology is a valid and valuable form of biology that has been difficult to investigate in the past owing to a lack of good technology. Atomic force microscopy (AFM) of DNA has been used to investigate DNA condensation for gene therapy, DNA mapping and sizing, and a few applications to cancer research and to nanotechnology. Some of the most exciting new applications for atomic force microscopy of DNA involve pulling on single DNA molecules to obtain measurements of single-molecule mechanics and thermodynamics.

Despite its remarkable materials properties, the structure of spider dragline silk has remained unsolved. Results from two probe microscopy techniques provide new insights into the structure of spider dragline silk. A soluble synthetic protein from dragline silk spontaneously forms nanofibers, as observed by atomic force microscopy. These nanofibers have a segmented substructure. The segment length and amino acid sequence are consistent with a slab-like shape for individual silk protein molecules. The height and width of nanofiber segments suggest a stacking pattern of slab-like molecules in each nanofiber segment. This stacking pattern produces nano-crystals in an amorphous matrix, as observed previously by NMR and x-ray diffraction of spider dragline silk. The possible importance of nanofiber formation to native silk production is discussed. Force spectra for single molecules of the silk protein demonstrate that this protein unfolds through a number of rupture events, indicating a modular substructure within single silk protein molecules. A minimal unfolding module size is estimated to be around 14 nm, which corresponds to the extended length of a single repeated module, 38 amino acids long. The structure of this spider silk protein is distinctly different from the structures of other proteins that have been analyzed by single-molecule force spectroscopy, and the force spectra show correspondingly novel features.

The atomic force microscope (AFM) was used to assay the extent of DNA condensation in ∼100 different complexes of DNA with polylysine (PL) or PL covalently attached to the glycoproteins asialoorosomucoid (AsOR) or orosomucoid (OR). The best condensation of DNA was obtained with 10 kDa PL covalently attached to AsOR, at a lysine:nucleotide (Lys:nt) ratio of 5:1 or higher. These conditions produce large numbers of toroids and short rods with contour lengths of 300–400 nm. Some DNA condensation into shortened thickened structures was seen with 10 kDa PL attached to AsOR at Lys:nt ratios of 1.6:1 and 3:1. Some DNA condensation was also seen with 4 kDa PL at Lys:nt ratios of 3:1 and higher. Little DNA condensation was seen with PL alone or with PL convalently attached to OR at Lys:nt ratios up to 6:1. AsOR-PL enhanced gene expression in the mouse liver ∼10- to 50-fold as compared with PL alone.