DNA Intercalation Facilitates Efficient DNA-Targeted Covalent Binding of Phenanthriplatin.

Phenanthriplatin, a monofunctional anticancer agent derived from cisplatin, shows significantly more rapid DNA covalent-binding activity compared to its parent complex. To understand the underlying molecular mechanism, we used single-molecule studies with optical tweezers to probe the kinetics of DNA-phenanthriplatin binding as well as DNA binding to several control complexes. The time-dependent extensions of single λ-DNA molecules were monitored at constant applied forces and compound concentrations, followed by rinsing with a compound-free solution. DNA-phenanthriplatin association consisted of fast and reversible DNA lengthening with time constant τ ≈ 10 s, followed by slow and irreversible DNA elongation that reached equilibrium in ∼30 min. In contrast, only reversible fast DNA elongation occured for its stereoisomer  trans-phenanthriplatin, suggesting that the distinct two-rate kinetics of phenanthriplatin is sensitive to the geometric conformation of the complex. Furthermore, no DNA unwinding was observed for pyriplatin, in which the phenanthridine ligand of phenanthriplatin is replaced by the smaller pyridine molecule, indicating that the size of the aromatic group is responsible for the rapid DNA elongation. These findings suggest that the mechanism of binding of phenanthriplatin to DNA involves rapid, partial intercalation of the phenanthridine ring followed by slower substitution of the adjacent chloride ligand by, most likely, the N7 atom of a purine base. The cis isomer affords the proper stereochemistry at the metal center to facilitate essentially irreversible DNA covalent binding, a geometric advantage not afforded by trans-phenanthriplatin. This study demonstrates that reversible DNA intercalation provides a robust transition state that is efficiently converted to an irreversible DNA-Pt bound state.

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

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.

A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps.

Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [µ-C4(cpdppz)2(phen)4Ru2](4+), which consists of two Ru(phen)2dppz(2+) moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins.

Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation.

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [µ-dppzip(phen)4Ru2](4+) (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)2dppz) and the distal subunit (Ru(phen)2ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δxeq, and the dynamic DNA structural deformations during ligand association xon and dissociation xoff. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a Kd of 27 ± 3 nM for (Δ,Λ)-Piz to a Kd of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δxeq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and xon from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited xoff changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics.

Strong DNA deformation required for extremely slow DNA threading intercalation by a binuclear ruthenium complex.

DNA intercalation by threading is expected to yield high affinity and slow dissociation, properties desirable for DNA-targeted therapeutics. To measure these properties, we utilize single molecule DNA stretching to quantify both the binding affinity and the force-dependent threading intercalation kinetics of the binuclear ruthenium complex Δ,Δ-[µ-bidppz-(phen)4Ru2]4+ (Δ,Δ-P). We measure the DNA elongation at a range of constant stretching forces using optical tweezers, allowing direct characterization of the intercalation kinetics as well as the amount intercalated at equilibrium. Higher forces exponentially facilitate the intercalative binding, leading to a profound decrease in the binding site size that results in one ligand intercalated at almost every DNA base stack. The zero force Δ,Δ-P intercalation Kd is 44 nM, 25-fold stronger than the analogous mono-nuclear ligand (Δ-P). The force-dependent kinetics analysis reveals a mechanism that requires DNA elongation of 0.33 nm for association, relaxation to an equilibrium elongation of 0.19 nm, and an additional elongation of 0.14 nm from the equilibrium state for dissociation. In cells, a molecule with binding properties similar to Δ,Δ-P may rapidly bind DNA destabilized by enzymes during replication or transcription, but upon enzyme dissociation it is predicted to remain intercalated for several hours, thereby interfering with essential biological processes.

Variable-force microscopy for advanced characterization of horizontally aligned carbon nanotubes.

Atomic force microscopy (AFM) performed with variable-force imaging was recently demonstrated to be an accurate method of determining the diameter and number of sidewalls of a carbon nanotube (CNT). This AFM technique provides an alternative to transmission electron microscopy (TEM) when TEM imaging is not possible due to substrate thickness. We have used variable-force AFM to characterize horizontally aligned CNTs grown on ST-cut quartz. Our measurements reveal new aspects of horizontally aligned growth that are essential for enhancing the performance of CNT-based devices as well as understanding the growth mechanism. First, previously reported optimal growth conditions produce a large spread in CNT diameters and a significant fraction of double-walled CNTs. Second, monodispersity is significantly improved when growth temperature is reduced. Third, CNTs with diameters up to 5 nm align to the substrate, suggesting the interaction between CNTs and the quartz lattice is more robust than previously reported.

DNA intercalation optimized by two-step molecular lock mechanism.

The diverse properties of DNA intercalators, varying in affinity and kinetics over several orders of magnitude, provide a wide range of applications for DNA-ligand assemblies. Unconventional intercalation mechanisms may exhibit high affinity and slow kinetics, properties desired for potential therapeutics. We used single-molecule force spectroscopy to probe the free energy landscape for an unconventional intercalator that binds DNA through a novel two-step mechanism in which the intermediate and final states bind DNA through the same mono-intercalating moiety. During this process, DNA undergoes significant structural rearrangements, first lengthening before relaxing to a shorter DNA-ligand complex in the intermediate state to form a molecular lock. To reach the final bound state, the molecular length must increase again as the ligand threads between disrupted DNA base pairs. This unusual binding mechanism results in an unprecedented optimized combination of high DNA binding affinity and slow kinetics, suggesting a new paradigm for rational design of DNA intercalators.