Mechanisms of small molecule–DNA interactions probed by single-molecule force spectroscopy

Abstract

There is a wide range of applications for non-covalent DNA binding ligands, and optimization of such interactions requires detailed understanding of the binding mechanisms. One important class of these ligands is that of intercalators, which bind DNA by inserting aromatic moieties between adjacent DNA base pairs. Characterizing the dynamic and equilibrium aspects of DNA-intercalator complex assembly may allow optimization of DNA binding for specific functions. Single-molecule force spectroscopy studies have recently revealed new details about the molecular mechanisms governing DNA intercalation. These studies can provide the binding kinetics and affinity as well as determining the magnitude of the double helix structural deformations during the dynamic assembly of DNA–ligand complexes. These results may in turn guide the rational design of intercalators synthesized for DNA-targeted drugs, optical probes, or integrated biological self-assembly processes. Herein, we survey the progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms. We discuss briefly minor and major groove binding ligands, and then focus on intercalators, which have been probed extensively with these methods. Conventional mono-intercalators and bis-intercalators are discussed, followed by unconventional DNA intercalation. We then consider the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules.

DNA intercalation optimized by two-step molecular lock mechanism

“…4A), as described in Methods. The obtained equilibrium elastic properties of the saturated DNA-Pi complex, including its contour length, persistence length, and elastic modulus, are comparable to values of these parameters previously measured for threading intercalators2324. Figure 4A also shows the effects of aggregation on the DNA stretching curves, which can only be observed at high concentrations when holding DNA at very low initial extensions, comparable to bulk experimental conditions, which also observed aggregation15.…”

A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition

“…Figure 1b shows complex 1 and 2 both increase relative viscosity of DNA, confirming that both these compounds bind DNA by intercalation. Notably, the increase in relative viscosity of DNA on addition of 1 was significantly greater than that for either 2 or ethidium bromide (EB), indicating 1 induces structural distortion to DNA at a substantially greater level than mono-intercalation, and is therefore consistent with a multi-intercalative binding interaction33. In addition to these cell-free DNA binding studies, the phosphorescent metal to ligand charge-transfer (MLCT) excitation/emission wavelengths may provide a direct indication of cellular DNA binding and several RPCs function as DNA imaging probes1314.…”

“…The cytotoxic DNA-damaging anti-cancer drugs cisplatin and mitoxantrone (MX), an organic intercalator and topoisomerase II poison33, were included in parallel for comparison. These experiments indicate that both Ru(II) compounds impact cell proliferation, with 2 demonstrating greater potency than 1 towards both cancer cell lines and possessing comparable IC 50 values to cisplatin (Table 1).…”

“…Also of note are distinct differences in the cellular response to 1 compared to topoisomerase inhibitors such as the organic intercalators mitoxantrone and doxorubicin, where DSB pathway activation, G2/M cell-cycle arrest and extensive apoptosis are observed8, indicating fork stalling by 1 is unlikely to occur via this mechanism. One potential explanation is provided by the viscosity data, which indicate that complex 1 , but importantly not complex 2 , induces lengthening and unwinding of duplex DNA to a greater extent than standard intercalation, indicating 1 to be a multi-intercalator33, and induces significant structural distortion to duplex DNA upon binding. Combined with the steric bulk of 1 , we propose DNA multi-intercalation by 1 in cells presents a physical block to replication, either by the resultant duplex distortion presenting a physical barrier to fork progression or a more complex binding interaction occurring at the fork resulting in stabilisation.…”

Some new Ag(I), VO(II) and Pd(II) chelates incorporating tridentate imine ligand: Design, synthesis, structure elucidation, density functional theory calculations for DNA interaction, antimicrobial and anticancer activities and molecular docking studies

“…Metal chelates interact with DNA probably through a mode that includes hydrophobic or electrostatic interaction. As expressed previously, the HNAPd metal chelate has a replaceable water ligand in solution, which might be substituted with DNA molecules in solution . Likewise because of the expulsion of water ligand from the metal chelate in solution, the explored metal chelates would have a flat part in the middle.…”