Minor Groove Binding Mode Research Articles

Initial DNA Interactions of the Binuclear Threading Intercalator Λ,Λ-[μ-bidppz(bipy)4Ru2]4+: An NMR Study with [d(CGCGAATTCGCG)]2

Binuclear polypyridine ruthenium compounds have been shown to slowly intercalate into DNA, following a fast initial binding on the DNA surface. For these compounds, intercalation requires threading of a bulky substituent, containing one RuII, through the DNA base-pair stack, and the accompanying DNA duplex distortions are much more severe than with intercalation of mononuclear compounds. Structural understanding of the process of intercalation may greatly gain from a characterisation of the initial interactions between binuclear RuII compounds and DNA. We report a structural NMR study on the binuclear RuII intercalator Λ,Λ-B (Λ,Λ-[μ-bidppz(bipy)4Ru2]4+; bidppz=11,11′-bis(dipyrido[3,2-a:2′,3′-c]phenazinyl, bipy = 2,2′-bipyridine) mixed with the palindromic DNA [d(CGCGAATTCGCG)]2. Threading of Λ,Λ-B depends on the presence and length of AT stretches in the DNA. Therefore, the latter was selected to promote initial binding, but due to the short stretch of AT base pairs, final intercalation is prevented. Structural calculations provide a model for the interaction: Λ,Λ-B is trapped in a well-defined surface-bound state consisting of an eccentric minor-groove binding. Most of the interaction enthalpy originates from electrostatic and van der Waals contacts, whereas intermolecular hydrogen bonds may help to define a unique position of Λ,Λ-B. Molecular dynamics simulations show that this minor-groove binding mode is stable on a nanosecond scale. To the best of our knowledge, this is the first structural study by NMR spectroscopy on a binuclear Ru compound bound to DNA. In the calculated structure, one of the positively charged Ru2+ moieties is near the central AATT region; this is favourable in view of potential intercalation as observed by optical methods for DNA with longer AT stretches. Circular dichroism (CD) spectroscopy suggests that a similar binding geometry is formed in mixtures of Λ,Λ-B with natural calf thymus DNA. The present minor-groove binding mode is proposed to represent the initial surface interactions of binuclear RuII compounds prior to intercalation into AT-rich DNA.

An investigation of the photophysical properties of minor groove bound and intercalated DAPI through quantum-mechanical and spectroscopic tools

The fluorescent probe 4',6-diamidino-2-phenylindole (DAPI) is a dye known to interact with polynucleotides in a non-univocal manner, both intercalation and minor groove binding modes being possible, and to specifically change its photophysical properties according to the different environments. To investigate this behavior, quantum-mechanical calculations using time-dependent density functional theory (TDDFT), coupled with polarizable continuum and/or atomistic models, were performed in combination with spectroscopic measurements of the probe in the different environments, ranging from a homogeneous solution to the minor groove or intercalation pockets of double stranded nucleic acids. According to our simulation, the electronic transition involves a displacement of the electron charge towards the external amidine groups and this feature makes the absorption energies very environment-sensitive while a much smaller sensitivity is seen in the fluorescence energies. Moreover, the calculations show that the DAPI molecule, when minor groove bound to the nucleic acid, presents both a reduced geometrical flexibility because of the rigid DNA pocket and a reduced polarization due to the very "apolar" microenvironment. All these effects can be used to better understand the observed enhancement of the fluorescence, which makes it an excellent marker for DNA.

DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes

Few transition metal complexes of tetradentate N2O2 donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL1/KHL2 have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL1/KHL2 are found to act as tetradentate ligands using N2O2 donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen (1O2) and superoxide anion radical (O2-) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method.

Benzothiadiazoles as DNA intercalators: Docking and simulation

AbstractSome organic dyes, derivatives of the 4‐(arylethynyl)‐7‐(4‐methoxyphenyl)‐2,1,3‐benzothiadiazoles (BTDs) bind strongly to double stranded DNA, exhibiting a strong increase in the fluorescence intensity. Due to this property, they were proposed as very sensitive probes for DNA detection and quantification. The details of the interaction mechanism, however, are unknown. In this work, we use docking and molecular dynamics simulations to study the interactions of these BTDs with DNA. We used a docking protocol where the receptor is a modified canonical oligonucleotide, with a gap, to sample intercalation and minor groove binding modes. Starting from several independent but energetically favorable docking poses, molecular dynamics simulations were carried out, investigating the ligand‐DNA interactions and the time evolution of the DNA structure. We observed, in agreement with experimental results, that the presence of the ethynyl spacer in the ligand stabilizes the intercalation binding mode, yielding strong interactions with DNA and large residence times. © 2012 Wiley Periodicals, Inc.

Designed Compounds for Recognition of 10 Base Pairs of DNA with Two AT Binding Sites

Short AT base pair sequences that are separated by a small number of GCs are common in eukaryotic parasite genomes. Cell-permeable compounds that bind effectively and selectively to such sequences present an attractive therapeutic approach. Compounds with linked, one or two amidine-benzimidazole-phenyl (ABP) motifs were designed, synthesized, and evaluated for binding to adjacent AT sites by biosensor-surface plasmon resonance (SPR). A surprising feature of the linked ABP motifs is that a set of six similar compounds has three different minor groove binding modes with the target sequences. Compounds with one ABP bind independently to two separated AT sites. Unexpectedly, compounds with two ABP motifs can bind strongly either as monomers or as cooperative dimers to the full site. The results are supported by mass spectrometry and circular dichroism, and models to explain the different binding modes are presented.

Multi-spectroscopic methods combined with molecular modeling dissect the interaction mechanisms of ractopamine and calf thymus DNA

The toxic interaction of ractopamine (RAC) with calf thymus DNA (ct DNA) was studied in vitro using multi-spectroscopic methods and molecular modeling methods. The hypochromic effect without a noticeable shift in UV-vis absorption indicated that the minor groove binding mode existed in the interaction between RAC and DNA. The fluorescence quenching of RAC was observed with the increasing addition of DNA and was proved to be the static quenching. The binding constant and the binding site sizes were 4.13 × 10(3) and 0.97, respectively. The thermodynamic calculation demonstrated that the hydrogen bond and van der Waals were main acting forces. This result further confirmed the existence of groove binding mode. Afterwards, we found another interaction mode, electrostatic binding mode through the fluorescence polarization, ionic effects and denatured DNA experiments. Circular dichroism spectroscopy (CD) was then employed to monitor the conformation changes of DNA. Molecular modeling studies illustrated the visual display of the binding mode and the detailed information of the H-bond.

Exploring the DNA binding mode of transition metal based biologically active compounds

Few novel 4-aminoantipyrine derived Schiff bases and their metal complexes were synthesized and characterized. Their structural features and other properties were deduced from the elemental analysis, magnetic susceptibility and molar conductivity as well as from mass, IR, UV–vis, 1H NMR and EPR spectral studies. The binding of the complexes with CT-DNA was analyzed by electronic absorption spectroscopy, viscosity measurement, and cyclic voltammetry. The interaction of the metal complexes with DNA was also studied by molecular modeling with special reference to docking. The experimental and docking results revealed that the complexes have the ability of interaction with DNA of minor groove binding mode. The intrinsic binding constants ( K b) of the complexes with CT-DNA were found out which show that they are minor groove binders. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the pUC19 DNA in the presence of AH 2 (ascorbic acid). Moreover, the oxidative cleavage studies using distamycin revealed the minor groove binding for the newly synthesized 4-aminoantipyrine derived Schiff bases and their metal complexes. Evaluation of antibacterial activity of the complexes against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and Klebsiella pneumoniae exhibited that the complexes have potent biocidal activity than the free ligands.

Interaction mechanism of 2-aminobenzothiazole with herring sperm DNA

The toxic interaction of 2-aminobenzothiazole (2-ABT) with herring sperm DNA (hs-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated that 2-ABT interacted with hs-DNA in a minor groove binding mode. The binding constant and the number of binding sites were 7.2×10 3 L mol −1 and 0.95, respectively. Circular dichroism spectroscopy (CD) was employed to measure the conformation change of hs-DNA in the presence of 2-ABT, which verified the minor groove binding mode. The molecular modeling results illustrated that 2-ABT tended to bind in the region of rich A–T base pairs through the hydrogen bond between A 18 and amino group of 2-ABT. Sequence specificity was confirmed by comparison on the interactions of 2-ABT with four kinds of bases. This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with biomacromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo.

Interaction between DNA and Microcystin-LR Studied by Spectra Analysis and Atomic Force Microscopy

Microcystin-LR (MC-LR) is one of the hepatotoxins produced by cyanobacteria in the eutrophicated fresh water. In this work, the minor groove binding mode of MC-LR to plasmid DNA was explored by using UV and fluorescence spectra, and the binding characteristics of MC-LR for plasmid DNA were calculated via the fluorescence quenching of ethidium bromide (EB) and mole ratio method. Furthermore, atomic force microscopy (AFM) was used to observe DNA morphology change in the presence of MC-LR. With the increasing concentration of MC-LR, circle DNA strands twined gradually to rod condensates. The possible reason for the condensation might be the masking of the electrostatic repulsion between DNA double strands by MC-LR. The present study might provide useful information for the pathopoiesis mechanism of MC-LR. More, because the condensation of DNA could affect the progresses of gene expression and protein transcription, it may implicate another trend to explore the nosogenesis of MC-LR.

Aggregation behavior of tetrakis(N, N′, N″, N‴-tetramethyl tetra-2,3-pyridino)porphyrazine copper(II) and its interaction with ct-DNA: a thermodynamic approach

The association behavior of tetrakis( N , N ′, N ″, N ‴-tetramethyl tetra-2,3-pyridino)porphyrazine copper(II) ([ Cu(II) 2,3- tmtppa ]4+) was investigated in aqueous solutions at 25 °C and various ionic strengths using optical absorption and resonance light scattering (RLS) spectroscopies. The results show that ([ Cu(II) 2,3- tmtppa ]4+) does not have any affinity for aggregation due to increasing of salt concentration and exists as monomers even in homogeneous aqueous solutions of high ionic strengths (more than 1 M NaCl ). Interaction of ([ Cu(II) 2,3- tmtppa ]4+) with calf thymus DNA has also been studied in 1 mM aqueous phosphate buffer of pH 7.0, by optical absorption and RLS spectroscopies, and thermal denaturation experiments. The appearance of hypochromicity of less than 10% and bathochromicity shift of Δλ ≤ 2 nm in UV-vis spectra of ([ Cu(II) 2,3- tmtppa ]4+), increasing of thermal melting point of DNA, and no change in RLS spectra of porphyrazine due to interaction with DNA, represent the minor outside groove binding mode without any stack aggregate formation. The thermodynamic of the binding of ([ Cu(II) 2,3- tmtppa ]4+)-DNA also has been studied. The binding constant (K) was obtained by analysis of the optical absorption spectra of the complex at various DNA concentrations using SQUAD software. The value of K was estimated to be 2.34 × 105 ± 0.06 M -1 at 25 °C. The thermodynamic parameters were calculated by van't Hoff equation. The enthalpy and entropy changes were 41.83 ± 3.28 kJ/mol and 242.08 ± 9.88 J/mol.K at 25 °C, respectively. The results indicate that the process is entropy-driven and suggest that hydrophobic interactions are the main driving forces for the complex formation. Increasing of the ionic strength due to addition of NaCl destabilized porphyrazine-DNA complexes, indicating the competition of Na + ions with porphyrazine complexes for occupation of minor groove binding sites.

Minor Groove Binding Compounds That Jump a GC Base Pair and Bind to Adjacent AT Base Pair Sites

Most A/T specific heterocyclic diamidine derivatives need at least four A/T base pairs for tight binding to the DNA minor groove. Addition of a GC base pair to A/T sequences typically causes a large decrease in binding constant. The ability to target biologically important sequences of DNA could be significantly increased if compounds that could recognize A/T sites with an intervening GC base pair could be designed. The kinetoplast DNA sequence of parasitic microorganisms, for example, contains numerous three A/T binding sites that are separated by a single G. A series of compounds were prepared to target the AAAGTTT sequence as a model system for discovery of "G-jumpers". The new synthetic compounds have two aromatic-amidine groups for A/T recognition, and these are connected through an oxy-methylene linker to cross the GC. CD experiments indicated a minor groove binding mode, as expected, for these compounds. T(max), surface plasmon resonance, and isothermal titration calorimetry experiments revealed 1:1 binding to the AAAGTTT sequence with an affinity that depends on compound structure. Benzimidazole derivatives gave the strongest binding and had generally good solution properties. The binding affinities to the classical AATT sequence were similar to that for AAAGTTT for these extended compounds, but binding was weaker to the AAAGCTTT sequence with two intervening GC base pairs. Binding to both AAAGTTT and AATT was enthalpy driven for strong binding benzimidazole derivatives.

The promotion of secondary structures in single-stranded DNA by drugs that bind toduplex DNA: an atomic force microscopy study

We study the behavior of single-stranded DNA (ssDNA) in the presence of well-knowndrugs with either an intercalating binding mode, such as daunorubicin, actinomycin D, andchloroquine, or a minor groove binding mode, such as netropsin and berenil, by atomicforce microscopy (AFM). At very low salt conditions, ssDNA molecules adopt anunstructured conformation without secondary structures. We observe that under theseconditions additions of drugs that bind to double-stranded DNA (dsDNA) promote theformation of secondary structures in ssDNA. Furthermore, with an increase ofconcentration of the drugs, the extension as well as the thermal stabilization of thesehairpins was observed.

Novel Amidino-Substituted Thienyl- and Furylvinylbenzimidazole: Derivatives and Their Photochemical Conversion into Corresponding Diazacyclopenta[c]fluorenes. Synthesis, Interactions with DNA and RNA, and Antitumor Evaluation. 4

Synthesis of novel nonfused amidino-substituted thienyl- and furylvinylbenzimidazole: derivatives and their photochemical cyclization into corresponding diazacyclopenta[ c]fluorenes is described. All studied compounds showed prominent growth inhibitory effect. The fused compounds showed stronger activity than nonfused ones, whereby imidazolyl-substituted compound 11 proved to be the most active one. Besides, it induced strong G2/M arrest of the cell cycle followed by drastic apoptosis, which is in accordance with the DNA intercalative binding mode determined by the spectroscopic studies. Nonfused derivatives induced strong S phase arrest of the cell cycle followed by apoptosis that together with DNA minor groove binding mode pointed to topoisomerase I inhibition. In addition, all nonfused compounds revealed pronounced selectivity toward tumor cells in comparison with nontumor cells. On the basis of the presented results, both nonfused and fused thiophene-containing imidazolyl derivatives should be considered as promising lead compounds for further investigation.

Amidine Analogues of Melphalan: Synthesis, Cytotoxic Activity, and DNA Binding Properties

Design, synthesis, and cytotoxic activity of amidine derivatives of melphalan are described and structure-activity relationships are discussed. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 human breast cancer cells demonstrated that these compounds were more active than melphalan. Data from the ethidium displacement assay showed that these compounds were able to bind in the minor groove-binding mode in AT sequences of DNA. The cytotoxic properties of the amidine analogues of melphalan towards cultured human breast cancer cells correlate with topoisomerase II inhibitory properties but not with DNA-binding properties.

FUNCTIONAL EVALUATION OF ELLIPTICAL TRAINER WITH HUMAN MOTION ANALYSIS

CA–TAT, a novel peptide analog, was modified at the N-terminus of TAT (47–57), the cell-penetrating peptide transacting activator of transcription, by attaching cecropin A (1–7). CA–TAT, TAT (47–57), and cecropin A (1–7) were synthesized using standard Fmoc solid-phase peptide synthesis procedures, purified using reversed-phase high performance liquid chromatography (RP–HPLC), and characterized using ESI–MS. CA–TAT demonstrated antibacterial activities against bacteria with low hemolysis (MHC > 128 μM). The minimum inhibitory concentration (MIC) values of CA–TAT were in the range of 1–16 μM, which completely inhibited both gram-positive and gram-negative bacteria. The interactions between CA–TAT or TAT (47–57) and calf thymus DNA (ct-DNA) were investigated using multi-spectroscopic techniques and viscometry. The results showed that both CA–TAT and TAT (47–57) can interact with DNA via the minor groove-binding mode, and binding constant was calculated to be 2.83 × 105 L mol−1 at 310 K, which is lower than that with the classical intercalation binder ethidium bromide (EB). Compared with TAT (47–57) or cecropin A (1–7), CA–TAT combined with DNA much closer. The study results suggest that CA–TAT can be used as a novel antibacterial peptide in the development of new antibiotics because of its antibacterial activity that targets intracellular DNA.

The dicationic derivatives of DBTAA: Interactions with DNA/RNA and antiproliferative effects on human cell lines

Three dibenzotetraaza[14]annulenes non-covalently interacted with double-stranded DNA and RNA by mixed minor groove and/or intercalative binding mode. Observed interactions were strongly dependent on the steric exposure of positive charges and the length of the linkers of studied compounds as well as on the secondary structure and basepair composition of DNA/RNA. Compound 2 showed pronounced selectivity toward dA–dT-rich sequences and binding mode switch from dominant minor groove binding to ds-DNA to dominant intercalation into ds-RNA. Antiproliferative effect of studied compounds on human tumor and normal cell lines was in good agreement with the strength of observed interactions with DNA/RNA.

The β-Carboline Analog Mana-Hox Causes Mitotic Aberration by Interacting with DNA

Mana-Hox, an analog of beta-carbolines with anticancer activity, induces aberrant mitosis and delays mitotic exit. However, the cellular target is not known. In this study, we visualized the intracellular localization of Mana-Hox. Mana-Hox rapidly penetrated into cells (within 1 min) and concentrated on disorganized metaphase chromosomes after 13 hr of exposure. We demonstrated that Mana-Hox is a noncovalent DNA binder that can interact with DNA through intercalation and/or through minor groove binding. Furthermore, Mana-Hox also inhibits topoisomerase II relaxation activity in vitro, suggesting that Mana-Hox could perturb mitotic chromosome decatenation. Overall, Mana-Hox binding to DNA plays a critical role in the induction of aberrant mitosis and contributes to its anticancer activity.

Interaction of deoxyribonucleic acid with H1 histone in the unirradiated and gamma irradiated states in vitro

This study was undertaken in order to find out the effect of solvent composition on mode of interaction of H 1 histone with DNA, suitability of DNA, H 1 histone and DNA-H 1 complex as in vitro biological dosimeters and protection possibility of DNA by H 1 histone against ionizing radiation-induced damage. Analytical techniques were used for the study of complex formation. Gamma irradiation was used to deliver doses to the biopolymers and their complexes. Equilibrium dialysis method was used for complex formation. FBX dosimetry was done for the delivery of total dose and dose rate determination. DNA : H 1 complex formation ratio were : 4.17 x 10 - 9 : 4.652 x 10 - 6 ML - 1 in 10 - 3 M phosphate buffer; 4.17 x 10 - 9 : 1.163 x 10 - 6 ML - 1 in 0.1 M phosphate buffer and 2.085 x 10 - 9 : 9.304 x 10 - 7 ML - 1 in 0.15 M SSC buffer. Linearity in response with absorbed dose up to 50 Gy (dose rate 1.50 Gy/min) was observed with (i) DNA at 207 nm and 259 mn at 4.17 x 10 - 9 ML - 1 , (ii) with H 1 histone at 202 nm and 270 nm at 1.163 x 10 - 6 ML - 1 in 0.1 M phosphate buffer, pH 7.0, (iii) with DNA: H 1 at 206 nm and 260 nm in 0.1 M phosphate buffer, (iv) with DNA: H 1 at 226 nm and 260 nm in SSC buffer. A constant decrease in fluorescent intensity (Ex 280 nm, Em 338 nm) was observed for H 1 histone with increase in dose. The average percentage hyperchromicity in 0.1 M phosphate buffer was 20% for DNA, 11% for H 1 histone and 17% for DNA: H 1 complex. In 0.15 M SSC buffer, for the complex it was 14% for a dose of 50 Gy at a dose rate of 1.5 Gy/min. The degrees of unfolding of the chromophores for this complex with dose were different in different solvents. Complex formations are due to electrostatic, minor groove binding and intercalation modes and are solvent dependent. DNA, H 1 and DNA: H 1 complex can be used as in vitro biological dosimetes. Binding modes are responsible for protection of DNA by H 1 histone up to a dose of 50 Gy.

Interactions of mixed ligand ruthenium(II) complexes containing an amino acid and 1,10-phenanthroline with DNA

Mixed ligand ruthenium(II) complexes containing an amino acid (AA) and 1,10-phenanthroline (phen), i.e. [Ru(AA)(phen) 2] n+ ( n=1,2, AA=glycine (gly), l-alanine ( l-ala), l-arginine ( l-arg)) have been synthesized. The interactions of these complexes and [Ru(phen) 3] 2+ with DNA have been examined by absorption, luminescence, and circular dichroism spectroscopic methods. Absorption spectral properties revealed that [Ru(AA)(phen) 2] + (AA=gly, l-ala) interacted with CT-DNA by the electrostatic binding mode. [Ru( l-arg)(phen) 2] 2+ exhibited the greatest hypochromicity, red shift, and binding constant, indicating that this complex may partially intercalate into the base-pairs of DNA. These results were also suggested by luminescence spectroscopy. CD spectral properties have been examined to understand the detailed interactions of the ruthenium(II) complexes with artificial DNA. In the case of Δ-[Ru( l-arg)(phen) 2] 2+, the solution on adding [poly(dG-dC)] 2 exhibited two well-defined positive peaks, which the shorter and longer wavelength peaks were assigned as originating from the major and the minor groove binding modes, respectively. Then, the solution on adding [poly(dA-dT)] 2 exhibited only one positive peak, which was assigned as a peak corresponding to the minor groove binding mode.

Mode of drug binding to DNA determined by optical tweezers force spectroscopy

Optical tweezers were employed to investigate the effects of small DNA-binding molecules on the low-force (≤ 15 pN) stretching behaviour of single DNA molecules. As the canonical B-DNA helix is not perturbed in this force regime, the effects on DNA elasticity observed upon drug binding provide useful insight into how DNA-binding drugs may alter in vivo processes. In this study, the effects of agents with different DNA binding modes were analysed. DNA force—extension curves were recorded in the presence of netropsin, a purely minor groove-binding antibiotic drug, ethidium bromide, an intercalating fluorescent dye, and berenil, an antiprotozoal drug proposed to exhibit both intercalative and minor groove-binding modes. Applying an approximation of the worm-like chain model, which describes the low-force stretching behaviour, the results were analysed in terms of the DNA contour length and persistence length. From these single molecule studies it was observed that minor groove-binding and intercalating modes of DNA-binding could be distinguished based on changes to DNA elasticity.