Supercoiling-dependent DNA binding: quantitative modeling and applications to bulk and single-molecule experiments.

DNA stores our genetic information and is ubiquitous in applications, where it interacts with binding partners ranging from small molecules to large macromolecular complexes. Binding is modulated by mechanical strains in the molecule and can change local DNA structure. Frequently, DNA occurs in closed topological forms where topology and supercoiling add a global constraint to the interplay of binding-induced deformations and strain-modulated binding. Here, we present a quantitative model with a straight-forward numerical implementation of how the global constraints introduced by DNA topology modulate binding. We focus on fluorescent intercalators, which unwind DNA and enable direct quantification via fluorescence detection. Our model correctly describes bulk experiments using plasmids with different starting topologies, different intercalators, and over a broad range of intercalator and DNA concentrations. We demonstrate and quantitatively model supercoiling-dependent binding in a single-molecule assay, where we directly observe the different intercalator densities going from supercoiled to nicked DNA. The single-molecule assay provides direct access to binding kinetics and DNA supercoil dynamics. Our model has broad implications for the detection and quantification of DNA, including the use of psoralen for UV-induced DNA crosslinking to quantify torsional tension in vivo, and for the modulation of DNA binding in cellular contexts.

Targeting DNA junction sites by bis-intercalators induces topological changes with potent antitumor effects.

Targeting inter-duplex junctions in catenated DNA with bidirectional bis-intercalators is a potential strategy for enhancing anticancer effects. In this study, we used d(CGTATACG)2, which forms a tetraplex base-pair junction that resembles the DNA-DNA contact structure, as a model target for two alkyl-linked diaminoacridine bis-intercalators, DA4 and DA5. Cross-linking of the junction site by the bis-intercalators induced substantial structural changes in the DNA, transforming it from a B-form helical end-to-end junction to an over-wounded side-by-side inter-duplex conformation with A-DNA characteristics and curvature. These structural perturbations facilitated the angled intercalation of DA4 and DA5 with propeller geometry into two adjacent duplexes. The addition of a single carbon to the DA5 linker caused a bend that aligned its chromophores with CpG sites, enabling continuous stacking and specific water-mediated interactions at the inter-duplex contacts. Furthermore, we have shown that the different topological changes induced by DA4 and DA5 lead to the inhibition of topoisomerase 2 activities, which may account for their antitumor effects. Thus, this study lays the foundations for bis-intercalators targeting biologically relevant DNA-DNA contact structures for anticancer drug development.

Chromatin damage generated by DNA intercalators leads to degradation of RNA Polymerase II.

In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.

Diverse Chiral Nanotubes Assembled from Identical DNA Strands.

DNA nanotubes with controllable geometries hold a wide range of interdisciplinary applications. When preparing DNA nanotubes of varying widths or distinct chirality, existing methods require repeatedly designing and synthesizing specific DNA sequences, which can be costly and laborious. Here, we proposed an intercalator-assisted DNA tile assembly method which enables the production of DNA nanotubes of diverse widths and chirality using identical DNA strands. Through adjusting the concentration of intercalators during assembly, the twisting direction and extent of DNA tiles could be modulated, leading to the formation of DNA nanotubes featuring controllable widths and chirality. Moreover, through introducing additional intercalators and secondary annealing, right-handed nanotubes could be reconfigured into distinct left-handed nanotubes. We expect that this method could be universally applied to modulating the self-assembly pathways of various DNA tiles and other chiral materials, advancing the landscape of DNA tile assembly.

DOX-DNA Interactions on the Nanoscale: In Situ Studies Using Tip-Enhanced Raman Scattering.

Chemotherapeutic anthracyclines, like doxorubicin (DOX), are drugs endowed with cytostatic activity and are widely used in antitumor therapy. Their molecular mechanism of action involves the formation of a stable anthracycline-DNA complex, which prevents cell division and results in cell death. It is known that elevated DOX concentrations induce DNA chain loops and overlaps. Here, for the first time, tip-enhanced Raman scattering was used to identify and localize intercalated DOX in isolated double-stranded calf thymus DNA, and the correlated near-field spectroscopic and morphologic experiments locate the DOX molecules in the DNA and provide further information regarding specific DOX-nucleobase interactions. Thus, the study provides a tool specifically for identifying intercalation markers and generally analyzing drug-DNA interactions. The structure of such complexes down to the molecular level provides mechanistic information about cytotoxicity and the development of potential anticancer drugs.

Forced intercalation-induced light-up peptides as fluorogenic indicators for the HIV-1 TAR RNA-ligand assay.

Fluorescence indicators capable of binding to human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA are powerful tools for the exploratory studies of the identification of anti-HIV drug candidates. This work presents a new design strategy for fluorogenic indicators with a transactivator of transcription (Tat)-derived peptide based on the forced intercalation of thiazole orange (TO) dyes (FIT). The developed 9-mer FIT peptide (RKKRR-TO-RRR: named FiLuP) features the TO unit integrated onto a Dap (2,3-diaminopropionic acid) residue in the middle of the Tat peptide sequence; the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. This facilitates a significant light-up response (450-fold at λem = 541 nm, Φfree = 0.0057, and Φbound = 0.61) upon binding to TAR RNA. The response of FiLuP is highly selective to TAR RNA over other non-cognate RNAs, and FiLuP maintains strong binding affinity (Kd = 1.0 ± 0.6 nM). Significantly, in contrast to previously developed Tat peptide-based FRET probes, FiLuP is able to discriminate between "competitive" and "noncompetitive" inhibitors when used in the fluorescence indicator displacement (FID) assay. The FID assay under stringent screening conditions is also possible, enabling super-strong competitive binders toward TAR RNA to be sieved out.

From Intercalation to External Binding: Ru(II) Complexes with a Spiro Ligand for TAR RNA Selective Binding and HIV-1 Reverse Transcriptase Inhibition.

As a typical RNA virus, the genetic information on HIV-1 is entirely stored in RNA. The reverse transcription activity of HIV-1 reverse transcriptase (RT) plays a crucial role in the replication and transmission of the virus. Non-nucleoside RT inhibitors (NNRTIs) block the function of RT by binding to the RNA binding site on RT, with very few targeting viral RNA. In this study, by transforming planar conjugated ligands into a spiro structure, we convert classical Ru(II) DNA intercalators into a nonintercalator. This enables selective binding to HIV-1 transactivation response (TAR) RNA on the outer side of nucleic acids through dual interactions involving hydrogen bonds and electrostatic attraction, effectively inhibiting HIV-1 RT and serving as a selective fluorescence probe for TAR RNA.

Ligand Characterization and DNA Intercalation of Ru(II) Polypyridyl Complexes: A Local Vibrational Mode Study.

We investigated in this work ruthenium-ligand bonding across the RuN framework in 12 Ru(II) polypyridyl complexes in the gas phase and solution for both singlet and triplet states, in addition to their affinity for DNA binding through π-π stacking interactions with DNA nucleobases. As a tool to assess the intrinsic strength of the ruthenium-ligand bonds, we determined local vibrational force constants via our local vibrational mode analysis software. We introduced a novel local force constant that directly accounts for the intrinsic strength of the π-π stacking interaction between DNA and the intercalated Ru(II) complex. According to our findings, [Ru(phen)2(dppz)]2+ and [Ru(phen)2(11-CN-dppz)]2+ provide an intriguing trade-off between photoinduced complex excitation and the strength of the subsequent π-π stacking interaction with DNA. [Ru(phen)2(dppz)]2+ displays a small singlet-triplet splitting and a strong π-π stacking interaction in its singlet state, suggesting a favorable photoexcitation but potentially weaker interaction with DNA in the excited state. Conversely, [Ru(phen)2(11-CN-dppz)]2+ exhibits a larger singlet-triplet splitting and a stronger π-π stacking interaction with DNA in its triplet state, indicating a less favorable photoinduced transition but a stronger interaction with DNA postexcitation. We hope our study will inspire future experimental and computational work aimed at the design of novel Ru-polypyridyl drug candidates and that our new quantitative measure of π-π stacking interactions in DNA will find a general application in the field.

Isatin-indoloquinoxaline click adducts with a potential to overcome platinum-based drug-resistance in ovarian cancer.

Herein, a series of isatin tethered indolo[2,3-b]quinoxaline hybrids was synthesized by considering the pharmacophoric features of known DNA intercalators and topoisomerase II inhibitors. The anti-proliferative properties of the synthesized compounds were evaluated against ovarian cancer cell lines (SKOV-3 and Hey A8). Four of the compounds exhibited promising anti-proliferative activities, with one of them being 10-fold more potent than cisplatin against drug-resistant Hey A8 cells. Further investigations were carried out to determine the DNA intercalating affinities of the most active compounds as potential mechanisms for their anti-proliferative activities. ADMET in silico studies were performed to assess the physicochemical, pharmacokinetics, and toxicity parameters of active compounds. This study, to the best of our knowledge, is the first report on the potential of isatin-indoloquinoxaline hybrids as structural blueprints for the development of new DNA intercalators. Additionally, it explores their potential to circumvent platinum-based resistance in ovarian cancer.

Ultrafast spectroscopy study of DNA photophysics after proflavine intercalation.

Proflavine (PF), an acridine DNA intercalating agent, has been widespread applied as an anti-microbial and topical antiseptic agent due to its ability to suppress DNA replication. On the other hand, various studies show that PF intercalation to DNA can increase photogenotoxicity and has potential chances to induce carcinomas of skin appendages. However, the effects of PF intercalation on the photophysical and photochemical properties of DNA have not been sufficiently explored. In this study, the excited state dynamics of the PF intercalated d(GC)9 • d(GC)9 and d(AT)9 • d(AT)9 DNA duplex are investigated in an aqueous buffer solution. Under 267 nm excitation, we observed ultrafast charge transfer (CT) between PF and d(GC)9 • d(GC)9 duplex, generating a CT state with an order of magnitude longer lifetime compared to that of the intrinsic excited state reported for the d(GC)9 • d(GC)9 duplex. In contrast, no excited state interaction was detected between PF and d(AT)9 • d(AT)9. Nevertheless, a localized triplet state with a lifetime over 5 µs was identified in the PF-d(AT)9 • d(AT)9 duplex.

Pharmacophore-based, rationale design, and efficient synthesis of novel tetrahydrobenzo[b]thiophene candidates as potential dual Topo I/II inhibitors and DNA intercalators.

A series of tetrahydrobenzo[b]thiophene derivatives was designed and synthesized as dual topoisomerase (Topo) I/II inhibitors implicating potential DNA intercalation. Ethyl-2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene-4-carboxylate (1) was prepared by modification of the Gewald reaction procedure using a Fe2O3 nanocatalyst and then it was used as a building block for the synthesis of tetrahydrobenzo[b]thiophene candidates (2-14). Interestingly, compound 14 showed the best cytotoxic potential against hepatocellular, colorectal, and breast cancer cell lines (IC50 = 7.79, 8.10, and 3.53 µM), respectively, surpassing doxorubicin at breast cancer (IC50 = 4.17 µM). Meanwhile, the Topo I and II inhibition assay displayed that compound 3 could exhibit the best inhibitory potential among the investigated candidates (IC50 = 25.26 and 10.01 nM), respectively, in comparison to camptothecin (IC50 = 28.34 nM) and doxorubicin (IC50 = 11.01 nM), as reference standards. In addition, the DNA intercalation assay showed that compound 14 could display the best binding affinity with an IC50 value of 77.82 µM in comparison to doxorubicin (IC50 = 58.03 µM). Furthermore, cell cycle and apoptosis analyses described that compound 3 prompts the G1 phase arrest in michigan cancer foundation-7 cancer cells and increases the apoptosis ratio by 29.31% with respect to untreated cells (2.25%). Additionally, the conducted molecular docking assured the promising binding of the investigated members toward Topo I and II with potential DNA intercalation. Accordingly, the synthesized compounds could be treated as promising anticancer candidates for future optimization.

Structure and Intercalation of Cysteine-Asparagine-Serine Peptide into Montmorillonite as an Anti-Inflammatory Agent Preparation-A DFT Study.

Peptides are receiving significant attention in pharmaceutical sciences due to their applications as anti-inflammatory drugs; however, many aspects of their interactions and mechanisms at the molecular level are not well-known. This work explores the molecular structure of two peptides-(i) cysteine (Cys)-asparagine (Asn)-serine (Ser) (CNS) as a molecule in the gas phase and solvated in water in zwitterion form, and (ii) the crystal structure of the dipeptide serine-asparagine (SN), a reliable peptide indication whose experimental cell parameters are well known. A search was performed by means of atomistic calculations based on density functional theory (DFT). These calculations matched the experimental crystal structure of SN, validating the CNS results and useful for assignments of our experimental spectroscopic IR bands. Our calculations also explore the intercalation of CNS into the interlayer space of montmorillonite (MNT). Our quantum mechanical calculations show that the conformations of these peptides change significantly during intercalation into the confined interlayer space of MNT. This intercalation is energetically favorable, indicating that this process can be a useful preparation for therapeutic anti-inflammatory applications and showing high stability and controlled release processes.

Computational Assessment of a Dual-Action Ru(II)-Based Complex: Photosensitizer in Photodynamic Therapy and Intercalating Agent for Inducing DNA Damage.

A combined quantum-mechanical and classical molecular dynamics study of a recent Ru(II) complex with potential dual anticancer action is reported here. The main basis for the multiple action relies on the merocyanine ligand, whose electronic structure allows the drug to be able to absorb within the therapeutic window and in turn efficiently generate 1O2 for photodynamic therapy application and to intercalate within two nucleobases couples establishing reversible electrostatic interactions with DNA. TDDFT outcomes, which include the absorption spectrum, triplet states energy, and spin-orbit matrix elements, evidence that the photosensitizing activity is ensured by an MLCT state at around 660 nm, involving the merocyanine-based ligand, and by an efficient ISC from such state to triplet states with different characters. On the other hand, the MD exploration of all the possible intercalation sites within the dodecamer B-DNA evidences the ability of the complex to establish several electrostatic interactions with the nucleobases, thus potentially inducing DNA damage, though the simulation of the absorption spectra for models extracted by each MD trajectory shows that the photosensitizing properties of the complex remain unaltered. The computational results support that the anti-tumor effect may be related to multiple mechanisms of action.

Switching between DNA binding modes with a photo- and redox-active DNA-targeting ligand, part II: the influence of the substitution pattern.

A disulfide-functionalized photoactive DNA ligand is presented that enables the control of its DNA-binding properties by a combination of a photocycloaddition reaction and the redox reactivity of the sulfide/disulfide functionalities. In particular, the initially applied ligand binds to DNA by a combination of intercalation and groove-binding of separate benzo[b]quinolizinium units. The association to DNA is interrupted by an intramolecular [4 + 4] photocycloaddition to the non-binding head-to-head cyclomers. In turn, the subsequent cleavage of these cyclomers with dithiothreitol (DTT) regains temporarily a DNA-intercalating benzoquinolizinium ligand that is eventually converted into a non-binding benzothiophene. As a special feature, this sequence of controlled deactivation, recovery and internal shut-off of DNA-binding properties can be performed directly in the presence of DNA.

DNA major versus minor groove occupancy of monomeric and dimeric crystal violet derivatives. Toward structural correlations.

Six monomeric (1a-1f) and five dimeric (2a-2e) derivatives of the triphenylmethane dye crystal violet (CV) have been prepared. Evaluation of the binding of these compounds to CT DNA by competitive fluorescent intercalator displacement (FID) assays, viscosity experiments, and UV and CD spectroscopy suggest that monomeric derivative 1a and dimeric derivative 2d likely associate with the major groove of DNA, while dimeric derivatives 2a and 2e likely associate with the minor groove of DNA. Additional evidence for the groove occupancy assignments of these derivatives was obtained from ITC experiments and from differential inhibition of DNA cleavage by the major groove binding restriction enzyme BamHI, as revealed by agarose gel electrophoresis. The data indicate that major groove ligands may be optimally constructed from dye units containing a sterically bulky 3,5-dimethyl-N,N-dimethylaniline group; furthermore, the groove-selectivity of olefin-tethered dimer 2d suggests that stereoelectronic interactions (n â†’ π*) between the ligand and DNA are also an important design consideration in the crafting of major-groove binding ligands.

Modulating the chemo-mechanical response of structured DNA assemblies through binding molecules.

Recent advances in DNA nanotechnology led the fabrication and utilization of various DNA assemblies, but the development of a method to control their global shapes and mechanical flexibilities with high efficiency and repeatability is one of the remaining challenges for the realization of the molecular machines with on-demand functionalities. DNA-binding molecules with intercalation and groove binding modes are known to induce the perturbation on the geometrical and mechanical characteristics of DNA at the strand level, which might be effective in structured DNA assemblies as well. Here, we demonstrate that the chemo-mechanical response of DNA strands with binding ligands can change the global shape and stiffness of DNA origami nanostructures, thereby enabling the systematic modulation of them by selecting a proper ligand and its concentration. Multiple DNA-binding drugs and fluorophores were applied to straight and curved DNA origami bundles, which demonstrated a fast, recoverable, and controllable alteration of the bending persistence length and the radius of curvature of DNA nanostructures. This chemo-mechanical modulation of DNA nanostructures would provide a powerful tool for reconfigurable and dynamic actuation of DNA machineries.

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.

Mechanistic Insights into the Ligand-Induced Unfolding of an RNA G-Quadruplex.

The cationic porphyrin TMPyP4 is a well-established DNA G-quadruplex (G4) binding ligand that can stabilize different topologies via multiple binding modes. However, TMPyP4 can have both a stabilizing and destabilizing effect on RNA G4 structures. The structural mechanisms that mediate RNA G4 unfolding remain unknown. Here, we report on the TMPyP4-induced RNA G4 unfolding mechanism studied by well-tempered metadynamics (WT-MetaD) with supporting biophysical experiments. The simulations predict a two-state mechanism of TMPyP4 interaction via a groove-bound and a top-face-bound conformation. The dynamics of TMPyP4 stacking on the top tetrad disrupts Hoogsteen H-bonds between guanine bases, resulting in the consecutive TMPyP4 intercalation from top-to-bottom G-tetrads. The results reveal a striking correlation between computational and experimental approaches and validate WT-MetaD simulations as a powerful tool for studying RNA G4-ligand interactions.

Anomalous Enhancement of Electrochemical Charge Transfer by a Ru Complex Ion Intercalator.

We have found that the DNA intercalator [Ru(bpy)2DPPZ]2+ (bpy = 2,2'-bipyridine; DPPZ = dipyrido[3,2-a:2',3'-c]phenazine) causes an anomalous increase in charge transfer in electrochemical impedance spectroscopy (EIS). With a carbonaceous electrode and a 1 mM hexacyanoferrate (1 mM [Fe(CN)6]3- and 1 mM [Fe(CN)6]4-) mediator, we found that adding only 1 µM [Ru(bpy)2DPPZ]2+ greatly enhanced the charge transfer between the electrode and hexacyanoferrate mediator, independently of other electrolytes or buffer components. The effect started with a one millionth amount of hexacyanoferrate. Since [Ru(bpy)2DPPZ]2+ can intercalate with dsDNA, the effect is highly applicable for dsDNA detection or PCR monitoring. With further developments of this method, EIS sensors not requiring specific electrode modifications should be possible.

Electrochemical Impedimetric Real-Time Polymerase Chain Reactions Using Anomalous Charge Transfer Enhancement.

We developed a new electrochemical impedimetric method for the real-time detection of polymerase chain reactions (PCR) based on our recent discovery that the DNA intercalator, [Ru(bpy)2DPPZ]2+, anomalously enhances charge transfer between redox mediators, K4[Fe(CN)6]/K3[Fe(CN)6], and a carbon electrode. Three mM [Fe(CN)6]3-/4- and 5 µM [Ru(bpy)2DPPZ]2+ were added to the PCR solution, and electrochemical impedance spectroscopy (EIS) measurements were performed at each elongation heat cycle. The charge transfer resistance (Rct) was initially low due to the presence of [Ru(bpy)2DPPZ]2+ in the solution. As PCR progressed, amplicon dsDNA was produced exponentially, and intercalated [Ru(bpy)2DPPZ]2+ ions, which could be detected as a steep Rct, increased at specific heat cycles depending on the amount of template DNA. The Rct increase per heat cycle, ΔRct, showed a peak at the same heat cycle as optical detection, proving that PCR can be accurately monitored in real time by impedance measurement. This simple method will enable a cost-effective and portable PCR device.