Atomic force microscopy of biochemically tagged DNA.

Abstract

Small fragments of DNA of known length were made with the polymerase chain reaction. These fragments had biotin molecules covalently attached at their ends. They were subsequently labeled with a chimeric protein fusion between streptavidin and two immunoglobulin G-binding domains of staphyloccocal protein A. This tetrameric species was expected to bind up to four DNA molecules via their attached biotin moieties. The DNA-protein complex was deposited on mica and imaged with an atomic force microscope. The images revealed the protein chimera at the expected location at the ends of the strands of DNA as well as the expected dimers, trimers, and tetramers of DNA bound to a single protein.The atomic force microscope (AFM) (1, 2) is a derivative of the better known scanning tunneling microscope (STM) (3). The AFM images by measuring and maintaining a constant load force exerted by a sharp probe as it scans over the surface. This characteristic of the AFM makes it ideally suited for imaging nonconducting samples like biological molecules. Several groups have previously used the AFM to image both single-and double-stranded DNA/(4-13).The demonstrated ability ofthe AFM to image DNA allows microscopists the opportunity to reembark on a series of experiments begun more than 30 years ago. Microscopists at that time tried to apply the newly developed ability of the transmission electron microscope (TEM) to image metalshadowed DNA with the aim of mapping and sequencing this molecule. Attempts at sequencing DNA (14-16) with the TEM involved modifying DNA bases so that they would provide different, contrasting signals. Experiments designed to map DNA with the TEM involved both the imaging of sequence-specific proteins bound to the DNA (17) as well as the identification of probes hybridized to single-stranded DNA (18).The AFM, due to its high resolution and its ability to image DNA under conditions where the native structure will be retained, offers the hope of being able to improve upon the mapping and sequencing work done by these early microscopists. To sequence DNA, the AFM must be able to differentiate between the four types of nucleotide base pairs. Present images of single-stranded DNA (9) do not offer sufficient resolution to observe the functional groups that distinguish one nucleotide base from another. However, other researchers have reported the ability to distinguish between pyrimidines and purines (nucleotide bases with one and two rings) in two-dimensional surface layers with the STM (19). These results suggest that, in the future, the resolution of the STM/AFM (scanning probe) instruments may be sufficient to determine the sequence of the nucleotides of DNA (20,21).In this paper we describe the imaging of DNA fragments marked at specific locations with protein tags. These results show that the AFM has sufficient resolution to map DNA. In its simplest form, mapping involves the measurement of the physical distance between two points along the DNA. In the experiments reported here, we have demonstrated the ...

A study of fibrous long spacing collagen ultrastructure and assembly by atomic force microscopy

“…The grooves were typically 2 nm deep and 20 nm in width, and were continuous along the bands and the interband regions for all mature fibrils examined. It is important to note that the finite tip width means that the depth of these ridges may be an underestimate, whereas tip convolution effects mean that the width is an overestimate (Murray et al, 1993;Hansma et al, 1992).…”

Nanomechanics of Streptavidin Hubs for Molecular Materials

“…This approach is based on genetically fusing various protein building blocks to monomeric streptavidin and exploiting the propensity of streptavidin monomers to self-assemble themselves into stable tetramers. [14,15] …”

“…AFM images clearly revealed (I27 6 -SM) 4 structures in which streptavidin formed a central hub with four protruding I27 6 arms (Figure 1e). Based on protein gel analysis (Figure S1–S3) and AFM images, we conclude that SMs, functionalized with tandem I27 and SNase repeats, correctly assemble into uniform tetramer structures which also preserve their ability to bind biotins [15] (Figure 1d and Figure S4). …”

Observation of binding and polymerization of Fur repressor onto operator-containing DNA with electron and atomic force microscopes.

“…Furthermore, negative staining provides additional information about the structural organization of proteins bound to DNA. Due to electron-induced damage ofthe biological specimen with EM, the atomic force microscope (AFM) is now considered an interesting tool for high-resolution imaging of interacting biological macromolecules, especially DNA and proteins (10)(11)(12)(13)(14)(15).…”