Fluorescent Intercalator Displacement Research Articles

A Nanoplasmonic‐Fluorescent Ruler for Detection of Site‐Specific Protein Binding to Composite DNA of Multiple Sites

A gold or silver nanoparticles (AuNPs and AgNPs)‐supported fluorescent intercalator displacement (FID) assay, termed nanoplasmonic‐fluorescent ruler, for site‐specific detection of protein binding to composite DNA of multiple sites is constructed. A 20 nm, 100 nm AuNPs, or 80 nm AgNPs is introduced to one end of double‐stranded DNA (dsDNA). The dsDNA–AuNPs conjugates are saturated with a DNA intercalator, thiazole orange (TO). The distance‐determined fluorescent quenching and enhancement by mNPs give each intercalated TO its own contribution to the overall fluorescent ­intensity, according to their distance to the particles. Protein binding at a specific DNA site displaces the respective TO molecules, causing relative fluorescent intensity drop that is of a function of distance. Proteins Sp1 and ERα and their composite DNA, containing two identical Sp1 sites and one ERα site, are studied. With the smaller particle (i.e., 20 nm AuNPs) and larger particles (i.e., 100 nm AuNPs and 80 nm AgNPs) as quenching and enhancement ­profilers, respectively, it is possible to detect preferential binding of Sp1 to one of its sites, identify protein bound, and determine the cooperative binding of Sp1 and ERα where conventional FID fails unless a comprehensive set of DNA with respective protein sites being mutated is involved.

Promoting DNA molecules association by amphiphilic derivatives of 1,3-diazaadamantanes containing hydrophobic side chains

Earlier, a new class of compounds, amphiphilic derivatives of 1,3-diazaadamantanes, capable of facilitating the strand exchange in the system of short oligonucleotides, has been discovered. Longer hydrophobic side chains in 1,3-diazaadamantanes have been found to promote stronger acceleration of the reaction. In this study, the interaction of two 1,3-diazaadamantane derivatives containing different side chains with DNA was investigated using optical methods. Concentrations of micelle formation by the 1,3-diazaadamantanes, as well as the ranges of concentrations where the compounds/water mixtures exist in the form of true solutions, were determined based on the increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate probe. The affinities of 1,3-diazaadamantanes to DNA were determined with fluorescent intercalator displacement (FID) assay. A significant increase in the hydrodynamic volume of short DNA hairpins in complexes with 1,3-diazaadamantanes was revealed by the estimation of the fluorescence polarization of ethidium bromide probe bound in the hairpins. The intermolecular association of DNA hairpins upon binding with 1,3-diazaadamantanes was confirmed by Forster resonance energy transfer in an equimolar mixture of hairpins fluorescently labeled with Cy-3 or Cy5. In the study, the number of positive charges on 1,3-diazaadamantane derivatives that contain side chains of different lengths was demonstrated to affect their affinity to DNA, while longer hydrophobic side chains ensured more efficient interaction between the DNA duplexes that may facilitate DNA strand exchange.

ChemInform Abstract: Guanidine‐Equipped Thiacalix[4]arenes: Synthesis, Interaction with DNA and Aggregation Properties.

New tetrakis-guanidinium-containing p-tert-butylthiacalix[4]arene forms monodisperse nanoparticles with diameter 44 nm in the concentration range of 40–100 μm. Its association constant with model DNA was evaluated as pKa = 3.96 by fluorescent intercalator displacement assay.

A small molecule peptidomimetic that binds to c-KIT1 G-quadruplex and exhibits antiproliferative properties in cancer cells

A modular synthesis of l-proline derived peptidomimetics has been developed using the CuI catalyzed Huisgen cycloaddition between an azido prolinamide with pyridine and benzene dicarboxamide containing dialkynes. Förster Resonance Energy Transfer (FRET) melting assay provided an initial indication that the pyridyl analogue can stabilize the c-KIT1 quadruplex DNA. A competitive FRET-melting assay and Fluorescent Intercalator Displacement (FID) assay suggest that the pyridyl ligand shows excellent selectivity for c-KIT1 quadruplex over duplex DNA and other investigated G-quadruplexes. Molecular docking studies indicate that the pyridyl ligand can adopt unique conformations upon binding to c-KIT1 quadruplex due to the presence of intramolecular hydrogen bonds. The pyridyl ligand can perturb cell cycle progression and induce necrotic cell death of human hepatocellular liver carcinoma HepG2 cells.

Ru-indoloquinoline complex as a selective and effective human telomeric G-quadruplex binder

Indoloquinoline and its derivatives have been reported to be a kind of efficient G-quadruplex binder and have been found to interact preferentially to intramolecular G-quadruplex and inhibit telomerase activity in human K562 cells and SW620 cells. In contrast to indoloquinoline derivatives, much less is known about the metal complex based on indoloquinoline or its derivative. In this report, we studied the interaction of ruthenium complex [Ru(bpy)2(itatp)]2+ containing indoloquinoline moiety with human telomeric G-quadruplex DNA (Telo22) and c-myc G-quadruplex DNA (Pu27) by UV–visible (UV–Vis), fluorescence spectroscopy, fluorescent intercalator displacement (FID), thermal denaturation studies and CD spectroscopy. The results suggest that [Ru(bpy)2(itatp)]2+ displays a strong π–π stacking interaction with human telomeric G-quadruplex with a high binding constant (∼107M−1), but just exhibits moderate binding affinity to c-myc G-quadruplex, thus showing significant selectivity to human telomeric G-quadruplex. The CD titration results indicate that [Ru(bpy)2(itatp)]2+ could effectively convert Telo22 into antiparallel G-quadruplex conformation, while in the c-myc G-quadruplex case, instead of promoting Pu27 to fold into G-quadruplex, [Ru(bpy)2(itatp)]2+ destroys the parallel G-quadruplex structure of Pu27.

Assembly of Palladium(II) and Platinum(II) Metallo‐Rectangles with a Guanosine‐Substituted Terpyridine and Study of Their Interactions with Quadruplex DNA

Two novel [2+2] metallo-assemblies based on a guanosine-substituted terpyridine ligand (1) coordinated to palladium(II) (2 a) and platinum(II) (2 b) are reported. These supramolecular assemblies have been fully characterized by NMR spectroscopy, ESI mass spectrometry and elemental analyses. The palladium(II) complex (2 a) has also been characterized by single crystal X-ray diffraction studies confirming that the system is a [2+2] metallo-rectangle in the solid state. The stabilities of these [2+2] assemblies in solution have been confirmed by DOSY studies as well as by variable temperature (1)H NMR spectroscopy. The ability of these dinuclear complexes to interact with quadruplex and duplex DNA was investigated by fluorescent intercalator displacement (FID) assays, fluorescence resonance energy transfer (FRET) melting studies, and electrospray mass spectrometry (ESI-MS). These studies have shown that both these assemblies interact selectively with quadruplex DNA (human telomeric DNA and the G-rich promoter region of c-myc oncogene) over duplex DNA, and are able to induce dimerization of parallel G-quadruplex structures.

Guanidine-equipped thiacalix[4]arenes: synthesis, interaction with DNA and aggregation properties

New tetrakis-guanidinium-containing p-tert-butylthiacalix[4]arene forms monodisperse nanoparticles with diameter 44 nm in the concentration range of 40–100 μm. Its association constant with model DNA was evaluated as pKa = 3.96 by fluorescent intercalator displacement assay.

Synthesis and analysis of the anticancer activity of platinum(II) complexes incorporating dipyridoquinoxaline variants.

Eight platinum(II) complexes with anticancer potential have been synthesised and characterised. These complexes are of the type [Pt(I(L))(A(L))](2+), where I(L) is either dipyrido[3,2-f:2',3'-h]quinoxaline (dpq) or 2,3-dimethyl-dpq (23Me2dpq) and A(L) is one of the R,R or S,S isomers of either 1,2-diaminocyclohexane (SS-dach or RR-dach) or 1,2-diaminocyclopentane (SS-dacp or RR-dacp). The CT-DNA binding of these complexes and a series of other complexes were assessed using fluorescent intercalator displacement assays, resulting in unexpected trends in DNA binding affinity. The cytotoxicity of the eight synthesised compounds was determined in the L1210 cell line; the most cytotoxic of these were [Pt(dpq)(SS-dach)]Cl2 and [Pt(dpq)(RR-dach)]Cl2, with IC50 values of 0.19 and 0.80 μM, respectively. The X-ray crystal structure of the complex [Pt(dpq)(SS-dach)](ClO4)2·1.75H2O is also reported.

Spectroscopic investigation on the interaction of copper porphyrazines and phthalocyanine with human telomeric G-quadruplex DNA

The G-quadruplex DNA is a novel target for anticancer drug discovery and many scientific groups are investigating interaction of small molecules with G-quadruplex DNA to discover therapeutic agents for cancer. Here, interaction of a phthalocyanine (Cu(PcTs)) and two tetrapyridinoporphyrazines ([Cu(2,3-tmtppa)]4+ and [Cu(3,4-tmtppa)]4+) with Na+ and K+ forms of human telomeric G-quadruplex DNA has been investigated by spectroscopic techniques. The results indicated that interaction of the cationic porphyrazines is remarkably stronger than the anionic phthalocyanine and they presumably bind to the G-quadruplex DNA through end-stacking. Fluorescent intercalator displacement assay implied the displacement ability of the complexes with thiazole orange. In addition, circular dichroism spectra of both quadruplex forms converge to the Na+ isoform after binding to the porphyrazines. In conclusion, the porphyrazines as the complexes that bind to the G-quadruplex DNA, could be suitable candidates for further investigations about inhibition of telomerase enzyme.

A Platinum(II) Phenylphenanthroimidazole with an Extended Side-Chain Exhibits Slow Dissociation from ac-KitG-Quadruplex Motif

A series of three platinum(II) phenanthroimidazoles each containing a protonable side-chain appended from the phenyl moiety through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) were evaluated for their capacities to bind to human telomere, c-Myc, and c-Kit derived G-quadruplexes. The side-chain has been optimized to enable a multivalent binding mode to G-quadruplex motifs, which would potentially result in selective targeting. Molecular modeling, high-throughput fluorescence intercalator displacement (HT-FID) assays, and surface plasmon resonance (SPR) studies demonstrate that complex 2 exhibits significantly slower dissociation rates compared to platinum phenanthroimidazoles without side-chains and other reported G-quadruplex binders. Complex 2 showed little cytotoxicity in HeLa and A172 cancer cell lines, consistent with the fact that it does not follow a telomere-targeting pathway. Preliminary mRNA analysis shows that 2 specifically interacts with the ckit promoter region. Overall, this study validates 2 as a useful molecular probe for c-Kit related cancer pathways.

Abstract 4549: Use of molecular dynamic simulations to explore changes in conformation and sequence preference for DNA-binding biaryl polyamide structures.

Abstract As a class, minor-groove non-covalent binding small molecules generally show A/T selectivity. The lack of G/C selectivity is thought to relate to the wider minor groove in G/C regions and the presence of the exocyclic 2-amino groups of guanines which project into the minor groove and prevent multiple close van der Waals contacts with the G/C-rich minor groove. AT-selective structures such as distamycin and netropsin orientate in the minor groove in an “NH down” conformation, with their NH groups pointing into the minor groove to enhance the isohelicity of the structures and produce favourable van der Waal interactions with the groove floor below. Using a distamycin scaffold as a starting point we introduced biaryl building blocks in place of pyrroles to change the curvature and van der Waals interaction potential of this novel class of polyamides. We started by designing a library of fragments to span two DNA base pairs based on simple phenyl-substituted heterocycles of sufficient length. With each library member containing one heterocyclic and one carbocyclic component, sufficient diversification was achieved through modifications to, and substitutions within, both components. This allowed the production of libraries of building blocks possessing different curvatures and hydrogen-bonding abilities with the potential to complement functional groups and hydrogen bond donors and acceptors in the walls and floor of the minor groove. A fluorescent intercalator displacement (FID) assay was then used to select molecules with a preference for GC-rather than AT-rich sequences which identified the 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) motif. Molecular dynamics simulations were undertaken using the AMBER v11 software in implicit solvent over a time-scale of 20ns with the structures in both a “carbonyl-in” and “NH-in” conformation. This demonstrated that the MPB component within the biaryl polyamides undergoes a switch in orientation, with a “carbonyl down” conformation preferred which induces GC specificity. As well as occupying an idealised hydrogen-bonding distance between two side-by-side guanine residues, MPB units contained within the MPB biaryl polyamide structures showed superior hydrogen bonding interactions with GC compared to AT sequences, with each carbonyl of the MPB unit hydrogen bonding to a guanine residue. This was further supported by free-energy of binding calculations undertaken using AMBER-MMPBSA, where the MPB units showed a distinct preference for GC sequences (a difference of -13kcal/mol in favour of “carbonyl -in” over “NH-in” structures). This discovery has been applied to the design and synthesis of more-complex MPB-containing DNA minor-groove binding ligands with a high level of GC-selectivity and significant in vitro and in vivo antitumour activity. Citation Format: Paul J M Jackson, Colin M. James, Khondaker M. Rahman, David E. Thurston. Use of molecular dynamic simulations to explore changes in conformation and sequence preference for DNA-binding biaryl polyamide structures. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4549. doi:10.1158/1538-7445.AM2013-4549

The structure of anthracycline derivatives determines their subcellular localization and cytotoxic activity.

The cytotoxic activities and subcellular localizations of clinically used and synthetic analogues of the anthracycline family of chemotherapeutic agents were studied. The structures of the anthracycline derivatives affected their cytotoxicity and the time required for these compounds to exert cytotoxic effects on tumor cells. Fluorescent DNA intercalator displacement experiments demonstrated that there was no correlation between the DNA intercalation properties and the cytotoxicity of the studied anthracycline derivatives. Confocal microscopy experiments indicated that structural differences led to differences in subcellular localization. All studied anthracycline derivatives were observed in lysosomes, suggesting that this organelle, which is involved in several processes leading to malignancy, may contain previously unidentified molecular targets for these antitumor agents.

FDA-approved Drugs Selected Using Virtual Screening Bind Specifically to G-quadruplex DNA

Guanine-rich sequences found in telomeres and oncogene promoters have the ability to form G-quadruplex structures. In this paper we describe the use of a virtual screening assay to search a database of FDA-approved compounds for compounds with the potential to bind G-quadruplex DNA. More than 750 telomerase inhibitors were identified in a literature search as acting through G-quadruplex stabilization, and from evaluation of these compounds, theoretical models capable of discriminating new compounds that bind G-quadruplex DNA were developed. Six compounds predicted to bind to the G-quadruplex structure were tested for their ability to bind to the human telomeric DNA sequence. Prochloroperazine, promazine, and chlorpromazine stabilized the G-quadruplex structure as determined by fluorescence resonance energy transfer techniques. These compounds also bound to promoter sequences of oncogenes such as c-myc and K-ras. Amitriptyline, imipramine, and loxapine were less stabilizing but did bind to the G-quadruplex. The ability of prochloroperazine, promazine, and chlorpromazine to recognize G-quadruplex structures was confirmed using a fluorescent intercalator displacement assay, in which displacement of thiazole orange from G-quadruplex structures was demonstrated. Interestingly, these compounds exhibited selectivity for the G-quadruplex structure as all had poor affinity for the duplex sequence.

Photoswitchable DNA-binding properties of a photochromic spirooxazine derivative

An N-methylphenanthrolinium-annelated spirooxazine derivative 2(SO) whose DNA-intercalating properties are reversibly changed by a photochromic reaction was prepared. Upon irradiation at 350 nm the spirooxazine is transformed to the corresponding photomerocyanine 2(PM) that binds to DNA. After irradiation with visible light the spirooxazine 2(SO), which exhibits no significant DNA-binding properties, is regained. The association of 2(PM) with DNA was examined by CD and absorption spectroscopy, fluorescent intercalator displacement and viscometric titration.

51 Platinum(II) phenanthroimidazoles with “clicked” side chains as selective G-quadruplex DNA binders

G-quadruplexes (noncanonical secondary structures), have gained recognition as viable targets for chemotherapeutic drug design based on their ability to interfere with cancer cell proliferation. These DNA structures, held together by Hoogsteen hydrogen bonds, result from the folding of guanine (G)-rich DNA sequences in the presence of potassium or sodium cations.(Lane et al., 2008) G-quadruplex (G4)-forming sequences have been identified throughout the human genome, and depending on the sequence, different monomeric topologies prevail. These varying topologies present a means of specific targeting of one polymorph rather than all polymorphs. Two regions that have been highly investigated as G4 targets for small molecule drugs are the telomeres and the promoters of oncogenes. To date, both organic and inorganic compounds with varying degrees of efficacy in their targeting of G4 structures have been reported.(Georgiades et al., 2010; Monchaud & Teulade-Fichou, 2008) We have previously shown that Pt(II) phenanthroimidazole binders are good stabilizers of both intermolecular and intramolecular human-telomere-derived G4 motifs.(Castor et al., 2012; Kieltyka et al., 2008) Now, we focus on broadening our target to include those G4s derived from the c-myc and c-kit oncogene promoters due to their apparent ability in controlling gene expression through regulating transcription and translation. We hypothesized that the addition of side chains to our existing phenanthroimidazole core will enable our complexes to discriminate between different groove environments presented by topologically distinct G4s. Thus, we have taken our core and have appended “clicked”-amine containing side chains from the phenyl moiety of the phenylphenanthroimidazole. Through a high-throughput fluorescence intercalator displacement assay, circular dichroism, surface plasmon resonance, and molecular modeling, we have evaluated three complexes with G4s derived from human telomere, c-myc, and c-kit sequences and have shown enhanced binding of the complexes with the c-kit sequence and an unprecedented kinetic profile from SPR. We hope that this kinetic profile results in a long residence time while interacting with the c-kit promoter G-quadruplex, resulting in lower levels of c-kit mRNA in human glioblastoma cells and subsequent down-regulation of the gene expression.

A new light-scattering sensor for screening G-quadruplex stabilizers based on DNA-folding-mediated assembly of gold nanoparticles.

In this contribution, we constructed a biosensor for screening G-quadruplex stabilizers based on the assembly of gold nanoparticles functionalized with guanine-rich (G-rich) ssDNA using a light-scattering technique. We synthesized a series of metal-terpyridine complexes and investigated their affinity for quadruplex-DNA using the biosensor. Using the ratio of intensity of the light-scattering peak (I-I0)/I0, it can intuitively present the sequence of ability of stabilizing G-quadruplex DNA as follows: complex 1 > complex 3 > MTX > complex 2 > complex 5 > complex 6 > complex 7 > complex 4 > methyl green > TO > complex 8 > cisplatin. As our method allows the quantitative analysis of stabilizer affinity, EC50 values obtained are 4.8, 5.3, 6.1, 6.6, 6.7, 18.2, 20.2, 21.5, 32.2, 41.5 and 48.1 μM for complex 1, complex 3, MTX, complex 2, complex 5, complex 6, complex 7, complex 4, methyl green, TO and complex 8, respectively. The results have been verified by G-quadruplex fluorescent intercalator displacement (G4-FID) analysis. The proposed approach is a simple, convenient, intuitive and highly sensitive assay for screening G-quadruplex stabilizers. Our developed biosensor provides a promising tool for screening anticancer drugs.

A comparative study of the interaction of two structurally analogous ruthenium complexes with human telomeric G-quadruplex DNA

Two new polypyridine ligands and their corresponding ruthenium(II) complexes have been prepared and characterized. The interactions of both complexes with human telomere quadruplex DNA (both the antiparallel basket and the mixed-hybrid G-quadruplex) have been studied by circular dichroism (CD), CD melting, UV-visible (UV-Vis), fluorescent intercalator displacement (FID) assays and molecular docking studies. The results show that both complexes can stabilize G-quadruplexes DNA and two complexes show different binding affinity for different G-quadruplexes DNA. The 1:1 stoichiometry was confirmed in the buffered solutions by the UV-Vis spectrophotometer using Job's plot method and molecular docking studies. We have also investigated the interaction between the complexes and duplex DNA to gain some insight into the selectivity of the complexes for G-quadruplex structures. FID studies have shown that the complexes have a modest selectivity for G-quadruplex versus duplex DNA.

Cationic Pentaheteroaryls as Selective G‐Quadruplex Ligands by Solvent‐Free Microwave‐Assisted Synthesis

We report herein a solvent-free and microwaved-assisted synthesis of several water soluble acyclic pentaheteroaryls containing 1,2,4-oxadiazole moieties (1-7). Their binding interactions with DNA quadruplex structures were thoroughly investigated by FRET melting, fluorescent intercalator displacement assay (G4-FID) and CD spectroscopy. Among the G-quadruplexes considered, attention was focused on telomeric repeats together with the proto-oncogenic c-kit sequences and the c-myc oncogene promoter. Compound 1, and to a lesser extent 2 and 5, preferentially stabilise an antiparallel structure of the telomeric DNA motif, and exhibit an opposite binding behaviour to structurally related polyoxazole (TOxaPy), and do not bind duplex DNA. The efficiency and selectivity of the binding process was remarkably controlled by the structure of the solubilising moieties.

Fluorescent intercalator displacement replacement (FIDR) assay: determination of relative thermodynamic and kinetic parameters in triplex formation—a case study using triplex-forming LNAs

Triplex forming oligonucleotides (TFOs) are the most commonly used approach for site-specific targeting of double stranded DNA (dsDNA). Important parameters describing triplex formation include equilibrium binding constants (Keq) and association/dissociation rate constants (kon and koff). The ‘fluorescent intercalator displacement replacement’ (FIDR) assay is introduced herein as an operationally simple approach toward determination of these parameters for triplexes involving TC-motif TFOs. Briefly described, relative rate constants are determined from fluorescence intensity changes upon: (i) TFO-mediated displacement of pre-intercalated and fluorescent ethidium from dsDNA targets (triplex association) and (ii) Watson–Crick complement-mediated displacement of the TFO and replacement with ethidium (triplex dissociation). The assay is used to characterize triplexes between purine-rich dsDNA targets and TC-motif TFOs modified with six different locked nucleic acid (LNA) monomers, i.e. conventional and C5-alkynyl-functionalized LNA and α-L-LNA pyrimidine monomers. All of the studied monomers increase triplex stability by decreasing the triplex dissociation rate. LNA-modified TFOs form more stable triplexes than α-L-LNA-modified counterparts owing to slower triplex dissociation. Triplexes modified with C5-(3-aminopropyn-1-yl)-LNA-U monomer Z are particularly stable. The study demonstrates that three affinity-enhancing features can be combined into one high-affinity TFO monomer: conformational restriction of the sugar ring, expansion of the pyrimidine π-stacking surface and introduction of an exocyclic amine.

Tetrasubstituted Phenanthrolines as Highly Potent, Water‐Soluble, and Selective G‐Quadruplex Ligands

Small molecules capable of stabilizing the G-quadruplex (G4) structure are of interest for the development of improved anticancer drugs. Novel 4,7-diamino-substituted 1,10-phenanthroline-2,9-dicarboxamides that represent hybrid structures of known phenanthroline-based ligands have been designed. An efficient synthetic route to the compounds has been developed and their interactions with various G4 sequences have been evaluated by Förster resonance energy transfer (FRET) melting assays, fluorescent intercalator displacement (FID), electrospray ionization mass spectrometry (ESI-MS), and circular dichroism (CD) spectroscopy. The preferred compounds have high aqueous solubility and are strong and potent G4 binders with a high selectivity over duplex DNA; thus, they represent a significant improvement over the lead compounds. Two of the compounds are inhibitors of HeLa and HT1080 cell proliferation.