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Pair-wise interactions at the single-molecule level can be done with nanoprobing techniques, such as AFM force spectroscopy, optical tweezers, and magnetic tweezers. These techniques can be used to probe interactions between well-characterized assemblies of biomolecules, such as monomer-dimer, dimer-dimer, and trimer-monomer. An important step of these techniques is the proper assembly of dimers, trimers, and higher oligomers to enable the interactions to be probed. We have developed a novel approach in which a defined number of peptides are assembled along a flexible polymeric molecule that serves as a linear matrix, termed as flexible nanoarray (FNA). The construct is synthesized with the use of phosphoramidite chemistry (PA), in which non-nucleoside PA spacers and standard oligonucleotide synthesis are used to grow the polymeric chain with the desired length. The reactive sites are incorporated during FNA synthesis. As a result, the FNA polymer contains a set of predesigned reactive sites to which the peptides are covalently conjugated. We describe the protocol for the synthesis of FNA and the application of this methodology to measure the molecular interactions between amyloid peptides of monomer-monomer, monomer-dimer, and dimer-dimer.

This article describes sample preparation techniques for AFM imaging of DNA and protein-DNA complexes. The approach is based on chemical functionalization of the mica surface with aminopropyl silatrane (APS) to yield an APS-mica surface. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations, and in a broad range of pH. The chapter describes the methodologies for the preparation of APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein-DNA complexes.

Surface preparation is a key step for reliable and reproducible imaging of DNA and protein-DNA complexes with atomic force microscopy (AFM). This article describes the approaches for chemical functionalization of the mica surface. One approach utilizes 3-aminopropyl-trietoxy silane (APTES), enabling one to obtain a smooth surface termed AP-mica. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations and in a broad range of pH. Another method utilizes aminopropyl silatrane (APS) to yield an APS-mica surface. The advantage of APS-mica compared with AP-mica is the ability to obtain reliable and reproducible time-lapse images in aqueous solutions. The chapter describes the methodologies for the preparation of AP-mica and APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The applications are illustrated with a number of examples.

Despite their rather recent invention, atomic force microscopes are widely available commercially. AFM and its special modifications (tapping mode and noncontact operation in solution) have been successfully used for topographic studies of a large number of biological objects including DNA, RNA, proteins, cell membranes, and even whole cells. AFM was also successfully applied to studies of nucleic acids and various protein DNA complexes. Part of this success is due to the development of reliable sample preparation procedures. This chapter describes one of the approaches based on chemical functionalization of mica surface with 1-(3-aminopropyl) silatrane (APS). One of the most important properties of APS-mica approach is that the sample can be deposited on the surface in a wide range of ionic strengths, in the absence of divalent cations and a broad range of pH. In addition to imaging of dried sample, APS-mica allows reliable and reproducible time lapse imaging in aqueous solutions. Finally, APS mica is terminated with reactive amino groups that can be used for covalent and ionic attachment of molecules for AFM force spectroscopy studies. The protocols for the preparation of APS, functionalization with APS mica and AFM probes, preparation of samples for imaging in air and in aqueous solutions, and force spectroscopy studies are outlined. All these applications are illustrated with a few examples.