DNA Threading Intercalation Research Articles

Left versus right: Exploring the effects of chiral threading intercalators using optical tweezers

Small-molecule DNA-binding drugs have shown promising results in clinical use against many types of cancer. Understanding the molecular mechanisms of DNA binding for such small molecules can be critical in advancing future drug designs. We have been exploring the interactions of ruthenium-based small molecules and their DNA-binding properties that are highly relevant in the development of novel metal-based drugs. Previously we have studied the effects of the right-handed binuclear ruthenium threading intercalator ΔΔ-[μ-bidppz(phen)4Ru2]4+, or ΔΔ-P for short, which showed extremely slow kinetics and high-affinity binding to DNA. Here we investigate the left-handed enantiomer ΛΛ-[μ-bidppz(phen)4Ru2]4+, or ΛΛ-P for short, to study the effects of chirality on DNA threading intercalation. We employ single-molecule optical trapping experiments to understand the molecular mechanisms and nanoscale structural changes that occur during DNA binding and unbinding as well as the association and dissociation rates. Despite the similar threading intercalation binding mode of the two enantiomers, our data show that the left-handed ΛΛ-P complex requires increased lengthening of the DNA to thread, and it extends the DNA more than double the length at equilibrium compared with the right-handed ΔΔ-P. We also observed that the left-handed ΛΛ-P complex unthreads three times faster than ΔΔ-P. These results, along with a weaker binding affinity estimated for ΛΛ-P, suggest a preference in DNA binding to the chiral enantiomer having the same right-handed chirality as the DNA molecule, regardless of their common intercalating moiety. This comparison provides a better understanding of how chirality affects binding to DNA and may contribute to the development of enhanced potential cancer treatment drug designs.

Synthesis and biological evaluation of novel tetranuclear cyclopalladated complex bearing thiosemicarbazone scaffold ligand: Interactions with double‐strand DNA, coronavirus, and molecular modeling studies

AbstractThreading intercalators are a novel class of materials that carry two substituents along the diagonal positions of an aromatic ring. When bound to DNA, these substituents project out in DNA grooves. Tetranuclear complexes appear to be promising threading intercalators for developing therapeutics against cancer and viral infections that require high nucleic acid binding affinity. The objective of this work was to prepare the thiosemicarbazone scaffold ligand [4‐ClC6H4CHN=NC(S)NHPh] and tetranuclear cyclopalladated complex [Pd(4‐ClC6H4CHN=NC(S)NHPh)4] and to characterize the compounds by elemental analysis, 1D and 2D NMR, HRMS, and IR spectroscopy. The calf thymus DNA (CT‐DNA) binding properties of the compounds were investigated in vitro under simulated physiological conditions using UV–vis spectroscopy, emission spectral titration, methylene blue competitive binding, circular dichroism, DNA thermal denaturation, DNA binding, and coronavirus interactions using molecular simulation. The compounds showed cytotoxic effect against both human breast (MCF‐7) and colorectal (HCT116) cancer cells in a dose‐dependent manner. We demonstrated that the compounds are promising for DNA threading intercalation binders with large DNA binding constants on the order of 107 M−1 magnitude.

DNA threading intercalation of enantiopure [Ru(phen)2bidppz]2+ induced by hydrophobic catalysis.

The enantiomers of a novel mononuclear ruthenium(ii) complex [Ru(phen)2bidppz]2+ with an elongated dppz moiety were synthesized. Surprisingly, the complex showed no DNA intercalating capability in an aqueous environment. However, by the addition of water-miscible polyethylene glycol ether PEG-400, self-aggregation of the hydrophobic ruthenium(ii) complexes was counter-acted, thus strongly promoting the DNA intercalation binding mode. This mild alteration of the environment surrounding the DNA polymer does not damage or alter the DNA structure but instead enables more efficient binding characterization studies of potential DNA binding drugs.

Equilibrium and site selective analysis for DNA threading intercalation of a new phosphine copper(I) complex: Insights from X-ray analysis, spectroscopic and molecular modeling studies.

To clarify the interaction of phosphine copper(I) complex with DNA, our study reports the synthesis of a new phosphine copper(I) complex, along with a detailed analysis of the geometry characterization and its interaction with double-stranded DNA. The triclinic phase Cu(PPh3)2(L)(I) with a tetrahedral geometry was identified as the product of the reaction of copper(I) iodide with (E,E)-N,N'-1,2-Ethanediylbis[1-(3-pyridinyl)methanimine] ligand and triphenylphosphine by single-crystal X-ray analysis. Molecular interaction of the synthesized complex with the calf thymus deoxyribonucleic acid (ct-DNA) was investigated in the physiological buffer (pH7.4) by multi-spectroscopic approaches associated with a competitive displacement towards Hoechst 33258 and methylene blue (MB) as groove and intercalator probes. The fluorescence and UV/Vis results detected the formation of a complex-DNA adduct in the ground-state with a binding affinity in order of 104M-1, which is in keeping with both groove binders and intercalators. The thermodynamic parameters, ΔS0=-200.31±0.08cal/mol·K and ΔH0=-63.11±0.24kcal/mol, confirmed that the van der Waals interaction is the main driving force for the binding process. Moreover, the ionic strength and pH effect experiments demonstrated the electrostatic interactions between the complex and DNA is negligible. Analysis of the molecular docking simulation declared the flat (E,E)-N,N'-1,2-Ethanediylbis[1-(3-pyridinyl)methanimine] part of the complex was inserted between the sequential A…T/A…T base pairs, while the phosphine substituents were located in the groove, i.e. threading intercalation. Besides, the cytotoxicity of the complex against the MCF-7 human breast cancer cells was detected at IC50=10μg/mL.

Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation.

Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (Δ,Δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.

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.

Significance of Steric Bulk in DNA Threading Intercalation Revelaed through Force-Dependent Kinetics

DNA threading intercalation is a DNA binding mode, whereby a molecule must thread a bulky group through the DNA duplex to allow a planar central section to stack with the DNA base pairs. Molecules that bind to DNA via threading intercalation display incredibly slow association and dissociation kinetics. This is a result of the large duplex deformations required for the ligand to thread and unthread the DNA. While this binding mode has been investigated extensively, the specific molecular characteristics that affect the binding to DNA are still poorly understood. This study utilizes single molecule stretching with optical tweezers to elucidate the effects that the bulk and geometry of the threading moieties have on DNA binding. Single molecule optical tweezers experiments are a powerful tool in analyzing threading intercalation because they allow for direct quantification of both the equilibrium behavior and kinetics of DNA-ligand binding. The equilibrium binding profile of the binuclear ruthenium complex ΔΔ-[μ-bidppz(bipy)4Ru2]4+ (ΔΔ-B) was quantified in this study, revealing a dissociation constant on the same order of magnitude as its previously studied, bulkier sister molecule ΔΔ-[μ-bidppz(phen)4Ru2]4+ (ΔΔ-P). ΔΔ-B shows faster on rates compared to ΔΔ-P at low force, while both on rates converge to comparable values at high force. The off rates of ΔΔ-B show no force dependence, whereas the off rates of ΔΔ-P are strongly facilitated by force. These results suggest that in the absence of force that ΔΔ-B threads DNA faster and unthreads slower, making ΔΔ-B a more ideal DNA binding anticancer drug.

The Role of the Threading Moiety in DNA Threading Intercalation by Ruthenium Dimer Complexes

There is significant interest in ruthenium dimer complexes due to their novel DNA binding properties. The binuclear ruthenium complex ΔΔ-[μ-bidppz(bipy)4Ru2]4+ that we investigate in this study binds to DNA by threading intercalation, in which one of the bulky ruthenium moieties threads through the DNA base pair stack to reach its bound state. The extremely slow dissociation from DNA of this threading intercalator gives it properties needed for chemotherapy drugs, while also making it difficult to study in traditional bulk experiments. In this study we use optical tweezers in order to quantify the threading kinetics as well as the equilibrium behavior of this ruthenium dimer complex to understand how changing the ancillary ligands coordinated to ruthenium (threading moiety) affects the process of threading. To observe the slow threading process, the DNA was held at a constant force in the presence of the ligand and the DNA extension was measured as a function of time until the extension reached equilibrium. When the concentration of the ligand is increased during these constant force measurements, the total extension of the DNA will also increase until it becomes saturated at some concentration as all of the ligand binding sites are filled. These measurements of the equilibrium DNA extension allow for a full quantitative description of the binding kinetics and equilibrium behavior of the complex. The preliminary data suggests that the ΔΔ-[μ-bidppz(bipy)4Ru2]4+ complex with its bicyclic bipyridine ligand, shows significantly faster kinetics compared to the ΔΔ-[μ-bidppz(phen)4Ru2]4+, complex with a tricyclic phenanthroline ligand counterpart.

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.

Kinetics of DNA Threading Intercalation by a Rigid Ruthenium Complex Dimer

Compounds that can intercalate DNA by threading between melted bases serve as a model of therapeutic drugs for malignant tumors. An essential property of effective antitumor activity requires a drug candidate to exhibit a slow dissociation rate from the intercalation state of DNA-drug complex. In particular, previous bulk experiments reported a very slow dissociation rate for the rigid Ruthenium complex dimer ΔΔ-P (Δ,Δ-[µ-(11,11'-bidppz)(phen)4Ru2]4+). We examined the kinetics of ΔΔ-P threading intercalation into an individual λ-DNA molecule held between a micropipette and an optical trap over a concentration range of 2-100 nM. DNA extensions due to threading intercalation are measured at several concentrations for constant stretching forces of 10-60 pN. As a result, fractional binding is determined as a function of force and concentration from the time-dependent approach to equilibrium extension, which yields equilibrium binding affinity. We also obtain the force and concentration-dependent on and off rates for the threading reaction. Furthermore, these measurements allow us to obtain the force-dependent and zero force binding affinity. By fitting these rates to the expected exponential dependence on force, we are able to extract the equilibrium change in DNA extension due to a single intercalation event as well as the distance to the transition state from the bound and unbound equilibrium states. These results show that the DNA length must increase significantly both for association and dissociation of the ligand, which explains the extremely slow binding kinetics.

Finding at-DNA - Kinetic Recognition of Long Adenine-Thymine Stretches by Metal-Ligand Complexes

High selectivity for long AT sequences can be attained by kinetically controlled DNA threading intercalation by binuclear ruthenium(II) complexes. The rate of intercalation is strongly correlated to the number of consecutive AT basepairs, being up to 2500 times faster with an AT polymer compared to mixed-sequence DNA.

Mechanically Manipulating the DNA Threading Intercalation Rate

The dumbbell shaped binuclear ruthenium complex DeltaDelta-P requires transiently melted DNA in order to thread through the DNA bases and intercalate DNA. Because such fluctuations are rare at room temperature, the binding rates are extremely low in bulk experiments. Here, single DNA molecule stretching is used to lower the barrier to DNA melting, resulting in direct mechanical manipulation of the barrier to DNA binding by the ligand. The rate of DNA threading depends exponentially on force, consistent with theoretical predictions. From the observed force dependence of the binding rate, we demonstrate that only one base pair must be transiently melted for DNA threading to occur.

Kinetic Recognition of AT‐Rich DNA by Ruthenium Complexes

Finding AT tracts: High selectivity for long AT sequences can be attained through kinetically controlled DNA threading intercalation by binuclear ruthenium(II) complexes (see picture). The rate of intercalation is strongly correlated to the number of consecutive AT base pairs and is up to 2500-times higher with poly(dAdT)2 than with mixed-sequence DNA.