Background: Specific interactions between complementary strands of DNA and other molecules are central to the storage, retrieval and modification of information in biological systems. Although in ...many cases the basic structures of duplex DNA and the binding energetics have been well characterized, little information is available about the forces in these systems. These forces are of critical importance because they must be overcome, for example, by protein machines during transcription and repair. Recent developments in atomic force microscopy make possible direct measurements of such forces between the individual oligonucleotide strands that form DNA duplexes.
Results: We used the chemical force microscopy technique, in which oligonucleotides are covalently linked to the force microscope probe tip and the sample surface, to measure the elongation and binding forces of individual DNA duplexes. The separation forces between complementary oligonucleotide strands were found to be significantly larger than the forces measured between noncomplementary strands, and to be consistent with the unbinding of a single DNA duplex. With increasing applied force, the separation of complementary strands proceeded in a stepwise manner: B-form DNA was stretched, then structurally transformed to a stable form of DNA approximately twice the length of the B form, and finally separated into single-stranded oligonucleotides. These data provide a direct measurement of the forces required to elastically deform and separate double-stranded DNA into single strands.
Conclusions: Force microscopy provides a direct and quantitative measurement of the forces and energetics required to stretch and unbind DNA duplexes. Because the measurements can be carried out readily on synthetic oligonucleotides and in the presence of exogenous molecules, this method affords an opportunity for directly assessing the energetics of distorting and unbinding specific DNA sequences and DNA complexes. Such data could provide unique insights into the mechanistic steps following sequence-specific recognition by, for example, DNA repair and transcription factors.
Imaging the nanoscale distribution of specific chemical and biological sites on live cells is an important challenge in current life science research. In addition to imaging the surface topography of ...live cells, atomic force microscopy (AFM) is increasingly used to probe their chemical groups and biological receptors. In chemical force microscopy, AFM tips are modified with specific functional groups, thereby allowing investigators to probe chemical sites and their interactions on a scale of only ∼25 functional groups. In molecular recognition imaging, tips are functionalized with specific biomolecules, or samples labeled with immunogold particles, enabling researchers to localize specific receptors. Clearly, these nanoscale investigations provide new avenues in cellular biology and microbiology for elucidating the structure–function relationships of cell surfaces. In this chapter, we discuss the principles of these AFM modalities and their applications in life science research.
The structures of (3-aminopropyl)triethoxysilane (APTES), 4-aminothiophenol (4-ATP) and 4-mercaptopyridine (4-MP) self-assembled monolayers (SAMs) are studied by quantum mechanics in order to explain ...the force titration curves of these amino-group-terminated SAMs. The surface charges and electrostatic surface potentials derived from the ab initio calculations can give satisfactory explanations for the experimental results. We also propose a simple model to simulate the force titration process. The force between the tip and sample can be estimated according to the slope coefficient of the curve of energy versus distance. This curve can lead to a better understanding of the force titration curves of amino-group-terminated SAMs.
Chemical Force Microscopy (CFM) was used to study the electrochemical behavior of azobenzene-terminated self-assembled monolayers (SAMs). Both the gold-coated AFM tip and the gold-coated silicon ...substrate were modified with a 4-((N-(2′-mercapto-ethyl)-amino) carbonyl) azobenzene monolayer. For such a system, only when the reduction/oxidation reaction of azobenzene group occurs, the adhesion force between the tip and sample shows a drastic change, demonstrating the ability of CFM in monitoring the electrochemical transformation of an azobenzene group.
We prepared patterned self-assembled monolayers (SAMs) consisting of hexadecanethiol (16AT) and ferrocenyldodecanethiol (12FAT). The samples were characterized by scanning force microscopy (SFM), ...X-ray photoelectron spectroscopy (XPS), electrochemistry and contact angle measurements. Lateral force mode (LFM) of SFM shows image contrast even between surface regions of quite similar hydrophobicity. The 12FAT regions undergo irreversible chemical changes and become electrochemically inactive upon long exposure to the laboratory atmosphere. These chemical changes can be monitored by LFM, XPS, contact angle and electrochemistry. The LFM images of the exposed and contaminated samples show a reversed frictional contrast relative to the LFM images of the fresh samples and to the LFM images of the exposed but ethanol-rinsed sample. XPS and SFM data show that the 12FAT regions show more contamination than the 16AT regions. Based on these observations, the mechanism of the LFM image contrast is discussed and other driving forces, arising not only from differences in hydrophobicity but also from basic material properties such as elasticity, packing and contamination, are suggested.
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