An understanding of DNA−ligand interactions at the molecular level is important for the design of new drugs and probes that can recognize specific DNA sequences and structural motifs. Interestingly, determining the mode-of-binding of a DNA ligand is not always straightforward due to uncertainties inherent in traditional assays. We have recently reported an exciting new assay utilizing scanning force microscopy (SFM) that can discern whether a ligand binds to DNA by intercalative or nonintercalative modes [Coury et al. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12283−12286]. Visualization of individual DNA molecules by SFM and observation of ligand-induced lengthening provides direct evidence for intercalation. Metal complexes of polypyridyl ligands have been extensively studied as new probes of DNA structure and function because they exist as chiral molecules with the potential of enantioselective recognition of DNA. The binding mode of even the most widely studied of the members of this group, tris(o-phenanthroline)ruthenium(II) (Ru(phen)3 2+), remains somewhat controversial due in large part to its low binding affinity. We report here the use of Ru(phen)3 2+ as a test of our new assay toward the studies of weakly-binding ligands and to resolve the ambiguity surrounding the mode-of-binding of Δ and Λ-Ru(phen)3 2+. Experiments reported here reveal that the experimental conditions of our assay do not preclude the binding of Ru(phen)3 2+ to DNA and that NO lengthening occurs. Our findings are consistent with the view that Ru(phen)3 2+ binds to duplex nucleic acids through nonintercalative modes.

No abstract

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

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Single molecule mechanical testing is providing new insights into macromolecular behavior of complex native proteins including the energetics and dynamics of protein domain folding/unfolding and protein-ligand binding. The performance of atomic force microscopy (AFM) in single molecule mechanical measurements has distinct advantages over other force measuring devices. The very precise displacement measurements afforded protein molecules have relied largely on non-specific physisorption of the molecules to bare substrates by the AFM scanner enables examination of single molecules that are much smaller that those examined by other techniques (e.g. optical tweezers). Current AFM methods for investigation of single using the eponymous "smash and grab' technique (see FIG. 1). This involves bringing the tip into contact with the protein adsorbate followed by tip retraction at a controlled rate. However, the point of contact with the molecule and its orientation during loading is unknown. As a result, the observed length at release is related only to the point of contact and not to the structure of the macromolecule (see FIG. 2). Only the persistence length can be extracted from fits of either the freely jointed or worm-like chain models to the force-separation data.We envision a strategy for mechanical testing of proteins with well-defined attachments at the termini of the polypeptide chain to the tip and substrate surfaces (see FIG. 3). This strategy will provide a rapid and reliable method for analysis of the macromolecular properties of these biomolecules that is free of the artifacts and assumptions implicit in data acquisition with the "smash and grab" technique. We are examining the utility of molecular recognition between streptavidin and biotin to make contact at a known position on a single biomolecule. Biotinylated molecules are covalently immobilized onto a tip array whereas streptavidin is covalently attached to a tipless cantilever. This strategy enables replicate measurements on the same molecule. Force-separation curves obtained for tensile-loaded molecules are analyzed using both the freely-jointed and worm-like chain models. Our present efforts are devoted to correlating the mechanical properties with nucleotide sequence, assessing the impact of streptavidin unfolding during tensile loading of DNA, and extending this approach to elastin mimetic synthetic polypeptides.