Minor Groove Of Duplex DNA Research Articles

Substituting Inosine for Guanosine in DNA: Structural and Dynamic Consequences

Inosine differs from the guanosine nucleoside only by the absence of the N2 amino group. Both nucleosides also have similar electrostatic potentials. Therefore, substituting I for G has been used to probe various properties of nucleic acids and to facilitate the interpretation of binding studies. In particular, the absence of the amino group permits the assessment of its importance in the binding of ligands to the minor groove of duplex DNA. It has been known for some time that an I-C base pair is of lower stability than a regular G-C base pair, which needs to be considered when making DNA constructs containing inosine. However, it is generally assumed that both base pairs are structurally highly similar. To test this assumption in an identical sequence environment, we have determined the fine structure of two hairpin DNA substrates that differ only in the substitution of an I-C base pair for a G-C base pair. The structures have been solved using nuclear magnetic resonance (NMR) restraints in conjunction with Mardigras and molecular dynamics. The structural data are complemented with thermodynamic and dynamic data to get a comprehensive evaluation of the consequences of G-C vs I-C base pair substitutions. Our data show a strong similarity in the structures of the hairpins, but a significant difference in the melting temperatures, T m. This difference is also reflected in the drastically decreased base pair lifetime of 7.4 milliseconds compared to the G-C base pair lifetime of 155 milliseconds. The substitution of I-C for G-C is to probe for specific effect due to the amino group is satisfactory, as long as the lowered thermal stability and the drastically increased local dynamics are considered.

Cytotoxicity activity, in silico molecular docking, protein- and DNA-binding study of a new Ni(II) Schiff base complex

One new nickel(II) complex, [Ni(L)] (1), was synthesized from the Schiff base ligand derived from pyrrole-2-carboxaldehyde and 1,3-diaminopropane. Complex 1 was characterized by elemental analysis, IR, UV-Vis and ESI mass spectroscopy, cyclic voltammetry, and single-crystal X-ray structure analysis. Crystallographic results show that two Ni(II) monomeric moieties are present with similar structural features but with slightly different bond lengths and bond angles. The geometry around the Ni(II) center is distorted square planar. DNA-binding properties of complex 1 were well explored by employing UV-Vis and fluorescence spectral methods, cyclic voltammetry, and by viscosity measurements. Similarly the protein-binding study was studied by multispectroscopic techniques using both BSA and HSA. The cytotoxicity study of the compound has also been evaluated. Notably, the in vitro cytotoxicity of complex 1 on two cancer cell lines (AGS and A549) demonstrates that complex 1 has very good anticancer activity. MTT assay, cell-cycle analysis, and annexin-V assay have been performed to know the extent of effect of complex 1 as anticancer agent. Further, in silico molecular docking study revealed that the nickel(II) complex fits into the minor groove of duplex DNA by hydrophobic interaction with functional groups of B-DNA.

Pyrrolobenzodiazepines (PBDs) do not bind to DNA G-quadruplexes.

The pyrrolo[2,1-c][1,4] benzodiazepines (PBDs) are a family of sequence-selective, minor-groove binding DNA-interactive agents that covalently attach to guanine residues. A recent publication in this journal (Raju et al, PloS One, 2012, 7, 4, e35920) reported that two PBD molecules were observed to bind with high affinity to the telomeric quadruplex of Tetrahymena glaucoma based on Electrospray Ionisation Mass Spectrometry (ESI-MS), Circular Dichroism, UV-Visible and Fluorescence spectroscopy data. This was a surprising result given the close 3-dimensional shape match between the structure of all PBD molecules and the minor groove of duplex DNA, and the completely different 3-dimensional structure of quadruplex DNA. Therefore, we evaluated the interaction of eight PBD molecules of diverse structure with a range of parallel, antiparallel and mixed DNA quadruplexes using DNA Thermal Denaturation, Circular Dichroism and Molecular Dynamics Simulations. Those PBD molecules without large C8-substitutents had an insignificant affinity for the eight quadruplex types, although those with large π-system-containing C8-substituents (as with the compounds evaluated by Raju and co-workers) were found to interact to some extent. Our molecular dynamics simulations support the likelihood that molecules of this type, including those examined by Raju and co-workers, interact with quadruplex DNA through their C8-substituents rather than the PBD moiety itself. It is important for the literature to be clear on this matter, as the mechanism of action of these agents will be under close scrutiny in the near future due to the growing number of PBD-based agents entering the clinic as both single-agents and as components of antibody-drug conjugates (ADCs).

Synthesis of bis-1,2,3-triazolo-bridged unsymmetrical pyrrolobenzodiazepine trimers via ‘click’ chemistry and their DNA-binding studies

New conceivable synthetic approach for the construction of nitrogen-rich 1,2,3-triazolo-pyrrolo[2,1- c][1,4]benzodiazepine (TPBD, 3a– c) trimers has been developed. The first example of a bis-1,2,3-triazolo-bridged unsymmetrical PBD trimer has been successfully synthesized by employing a CuAAC type ‘click’ chemistry protocol. This efficient route generates tri-imine functionality in a single molecule. It has been envisaged that such tri-imine functionalities could bring in efficient interaction with DNA in a sequence-selective manner in the minor groove of duplex DNA. One of the representative analogues 3c has shown improved DNA-binding ability (Δ T m 23.7 °C) by thermal denaturation studies using CT-DNA and this data is also supported by molecular modeling (MD) studies.

Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι

Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase iota was measured and structures of the DNA polymerase iota-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase iota opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase iota incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase iota opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase iota extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase iota catalyzed reaction is well suited for N(2)-ethyl-Gua bypass. The structure of DNA polymerase iota with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase iota complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase iota polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase iota efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.

Interstrand DNA Cross-Links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources

Significant levels of the 1, N(2)-gamma-hydroxypropano-dG adducts of the alpha,beta-unsaturated aldehydes acrolein, crotonaldehyde, and 4-hydroxy-2E-nonenal (HNE) have been identified in human DNA, arising from both exogenous and endogenous exposures. They yield interstrand DNA cross-links between guanines in the neighboring C.G and G.C base pairs located in 5'-CpG-3' sequences, as a result of opening of the 1,N(2)-gamma-hydroxypropano-dG adducts to form reactive aldehydes that are positioned within the minor groove of duplex DNA. Using a combination of chemical, spectroscopic, and computational methods, we have elucidated the chemistry of cross-link formation in duplex DNA. NMR spectroscopy revealed that, at equilibrium, the acrolein and crotonaldehyde cross-links consist primarily of interstrand carbinolamine linkages between the exocyclic amines of the two guanines located in the neighboring C.G and G.C base pairs located in 5'-CpG-3' sequences, that maintain the Watson-Crick hydrogen bonding of the cross-linked base pairs. The ability of crotonaldehyde and HNE to form interstrand cross-links depends upon their common relative stereochemistry at the C6 position of the 1,N(2)-gamma-hydroxypropano-dG adduct. The stereochemistry at this center modulates the orientation of the reactive aldehyde within the minor groove of the double-stranded DNA, either facilitating or hindering the cross-linking reactions; it also affects the stabilities of the resulting diastereoisomeric cross-links. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease. Reduced derivatives of these cross-links are useful tools for studying their biological processing.

Metal‐Ion‐Mediated Tuning of Duplex DNA Binding by Bis(2‐(2‐pyridyl)‐1H‐benzimidazole)

Studies of double-stranded-DNA binding have been performed with three isomeric bis(2-(n-pyridyl)-1H-benzimidazole)s (n=2, 3, 4). Like the well-known Hoechst 33258, which is a bisbenzimidazole compound, these three isomers bind to the minor groove of duplex DNA. DNA binding by the three isomers was investigated in the presence of the divalent metal ions Mg(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+). Ligand-DNA interactions were probed with fluorescence and circular dichroism spectroscopy. These studies revealed that the binding of the 2-pyridyl derivative to DNA is dramatically reduced in the presence of Co(2+), Ni(2+), and Cu(2+) ions and is abolished completely at a ligand/metal-cation ratio of 1:1. Control experiments done with the isomeric 3- and 4-pyridyl derivatives showed that their binding to DNA is unaffected by the aforementioned transition-metal ions. The ability of 2-(2-pyridyl)benzimidazole to chelate metal ions and the conformational changes of the ligand associated with ion chelation probably led to such unusual binding results for the ortho isomer. The addition of ethylenediaminetetraacetic acid (EDTA) reversed the effects completely.

Binding of netropsin to several DNA constructs: Evidence for at least two different 1:1 complexes formed from an –AATT-containing ds-DNA construct and a single minor groove binding ligand

Isothermal titration calorimetry, ITC, has been used to determine the thermodynamics (Δ G, Δ H, and − TΔ S) for binding netropsin to a number of DNA constructs. The DNA constructs included: six different 20–22mer hairpin forming sequences and an 8-mer DNA forming a duplex dimer. All DNA constructs had a single –AT-rich netropsin binding with one of the following sequences, (A 2T 2) 2, (ATAT) 2, or (AAAA/TTTT). Binding energetics are less dependent on site sequence than on changes in the neighboring single stranded DNA (hairpin loop size and tail length). All of the 1:1 complexes exhibit an enthalpy change that is dependent on the fractional saturation of the binding site. Later binding ligands interact with a significantly more favorable enthalpy change (∂Δ H 1–2 from 2 to 6 kcal/mol) and a significantly less favorable entropy change (∂(− TΔ S 1–2)) from − 4 to − 9 kcal/mol). The ITC data could only be fit within expected experimental error by use of a thermodynamic model that includes two independent binding processes with a combined stoichiometry of 1 mol of ligand per 1 mol of oligonucleotide. Based on the biophysical evidence reported here, including theoretical calculations for the energetics of “trapping” or structuring of a single water molecule and molecular docking computations, it is proposed that there are two modes by which flexible ligands can bind in the minor groove of duplex DNA. The higher affinity binding mode is for netropsin to lay along the floor of the minor groove in a bent conformation and exclude all water from the groove. The slightly weaker binding mode is for the netropsin molecule to have a slightly more linear conformation and for the required curvature to be the result of a water molecule that bridges between the floor of the minor groove and two of the amidino nitrogens located at one end of the bound netropsin molecule.

A New Way To Detect Noncovalently Bonded Complexes of Biomolecules from Liquid Micro-Droplets by Laser Mass Spectrometry

A new version of laser mass-spectrometry is presented, which allows the quantitative analysis of specific biocomplexes in native solution. On-demand micro droplets, injected into vacuum, are irradiated by mid IR-laser pulses. Above a certain intensity threshold they explode due to the transmitted energy, setting free a fraction of the charged biomolecules which are then mass-analyzed. Amounts of analyte in the attomolar range may be detected with the ion intensity being linear over a wide range of molarity. Evidence is given that this method is soft, tolerant against various buffers, reflects properties of the liquid phase, and suitable for studying noncovalently bonded specific complexes. This is highlighted by results from antibiotics specifically binding into the minor groove of duplex DNA.

Thermodynamics of DNA Binding and Distortion by the Hyperthermophile Chromatin Protein Sac7d

Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66 degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 degrees C (Ka = 3.5(+/-1.6) x 10(6) M(-1)) and increased by nearly an order of magnitude from 10 degrees C to 80 degrees C. The enthalpy of binding was unfavorable at 25 degrees C, and decreased linearly from 5 degrees C to 60 degrees C. A positive binding heat at 25 degrees C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6 degrees C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 degrees C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 degrees C. In addition, an anomalous positive DeltaCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a Tm of 15-20 degrees C and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.

Mechanistic and anti-proliferative studies of two novel, biologically active bis-benzimidazoles

We have previously synthesised a number of novel head-to-head bis-benzimidazole derivatives that are structurally related to the fluorochrome, Hoechst 33258, and which possess strong affinity for A:T sites in the minor groove of duplex DNA. Initial studies revealed these compounds to exhibit potent antiproliferative activity against a range of ovarian cell lines and to inhibit transcription in an in vitro setting. In this study, we have examined their cellular behaviour in detail and have shown that two of these compounds (ABA13 and ABA833) potently inhibit the proliferation of a range of human tumour cell lines, and show some specificity towards breast carcinoma cell lines. In most of the cell lines investigated, ABA833 was the more potent of the two compounds. Flow cytometric analysis revealed that ABA13 and ABA833 (50–500 nM) induced an S phase block and increased the pre-G1 population in MCF-7 and MDA 468 human breast cancer cells. An increase in the pre-G1 population of RKO colon carcinoma cells was seen only at 500 nM with ABA833, reflecting the reduced sensitivity of this cell line to the bis-benzimidazoles in comparison to the breast cancer cell lines. Mechanistic studies revealed that neither ABA13 or ABA833 act as topoisomerase I (topo I) or topoisomerase II (topo II) poisons in plasmid or kinetoplast DNA (kDNA) relaxation assays, but both compounds do inhibit the catalytic activity of these enzymes. Drug uptake studies showed that reduced sensitivity of MCF-7adr and RKO cells compared with MCF-7 to both ABA13 and ABA833 correlated with a markedly reduced intracellular drug accumulation.

Specific interactions of distamycin with G-quadruplex DNA.

Distamycin binds the minor groove of duplex DNA at AT-rich regions and has been a valuable probe of protein interactions with double-stranded DNA. We find that distamycin can also inhibit protein interactions with G-quadruplex (G4) DNA, a stable four-stranded structure in which the repeating unit is a G-quartet. Using NMR, we show that distamycin binds specifically to G4 DNA, stacking on the terminal G-quartets and contacting the flanking bases. These results demonstrate the utility of distamycin as a probe of G4 DNA-protein interactions and show that there are (at least) two distinct modes of protein-G4 DNA recognition which can be distinguished by sensitivity to distamycin.

On the kinetics of distamycin binding to its target sites on duplex DNA.

Distamycin A is a well known polyamide antibiotic that can bind in the minor groove of duplex DNA primarily at AT-rich sequences both as a monomer or as a side-by-side antiparallel dimer. The association phase of the distamycin binding reaction has not been studied in either of its binding modes, because of the lack of an adequate UV or CD signal at the low concentrations needed to monitor the fast bimolecular reaction. We report a significant increase in fluorescence amplitude, accompanied by a small red shift, on binding distamycin to its specific target sites. This signal can be used to monitor drug binding in steady-state and time-resolved processes. Distamycin shows extremely fast association with the 1:1 binding site, with a bimolecular rate of 7 x 10(7) M(-1) small middle dots(-1) and also fairly rapid dissociation ( approximately 3 s(-1)). When DNA is in excess, there is a slow component in the association reaction whose rate decreases strongly with increasing DNA concentration. Binding of the drug to the 2:1 site occurs in two distinct steps: fast, sequential binding of each drug molecule to the DNA with a bimolecular rate comparable to that at the 1:1 site, followed by a slow ( approximately 4 s(-1)) equilibration to the final population. Dissociation from the 2:1 site is approximately 40-fold slower than from the 1:1 site. This study provides the groundwork for analysis of the binding kinetics of longer polyamides and covalently linked polyamides that have recently been shown to inhibit transcription in vivo.

Recent developments in sequence selective minor groove DNA effectors.

DNA is a well characterized intracellular target but its large size and sequential nature make it an elusive target for selective drug action. Binding of low molecular weight ligands to DNA causes a wide variety of potential biological responses. In this respect the main consideration is given to recent developments in DNA sequence selective binding agents bearing conjugated effectors because of their potential application in diagnosis and treatment of cancers as well as in molecular biology. Recent progress in the development of cross linked lexitropsin oligopeptides and hairpins, which bind selectively to the minor groove of duplex DNA, is discussed. Bis-distamycins and related lexitropsins show inhibitory activity against HIV-1 and HIV-2 integrases at low nanomolar concentrations. Benzoyl nitrogen mustard analogs of lexitropsins are active against a variety of tumor models. Certain of the bis-benzimidazoles show altered DNA sequence preference and bind to DNA at 5'CG and TG sequences rather than at the preferred AT sites of the parent drug. A comparison of bifunctional bizelesin with monoalkylating adozelesin shows that it appears to have an increased sequence selectivity such that monoalkylating compounds react at more than one site but bizelesin reacts only at sites where there are two suitably positioned alkylation sites. Adozelesin, bizelesin and carzelesin are far more potent as cytotoxic agents than cisplatin or doxorubicin. A new class of 1,2,9,9a-tetrahydrocyclo-propa[c]benz[e]indole-4-one (CBI) analogs i.e., CBI-lexitropsin conjugates arising from the latter leads are also discussed.A number of cyclopropylpyrroloindole (CPI) and CBI-lexitropsin conjugates related to CC-1065 alkylate at the N3 position of adenine in the minor groove of DNA in a sequence specific manner, and also show cytotoxicities in the femtomolar range. The cross linking efficiency of PBD dimers is much greater than that of other cross linkers including cisplatin, and melphalan. A new class of PBD-lexitropsin conjugates is also discussed. Certain functional models of the bleomycins (BLMs) show outstanding DNA cleavage activity comparable with that of and positionally distinct from natural BLM.

An Alteration in the Structure of the Minor Groove of Duplex DNA Induced by the Formation of an Intermolecular d(GA)n:d(GA)n ·d(TC)n Triplex

An Alteration in the Structure of the Minor Groove of Duplex DNA Induced by the Formation of an Intermolecular d(GA)n:d(GA)n ·d(TC)n Triplex

Anthraquinone photonucleases: a surprising role for chloride in the sequence-neutral cleavage of DNA and the footprinting of minor groove-bound ligands.

Irradiation of water-soluble anthraquinone (AQ) reagents in the presence of chloride ions results in the spontaneous, sequence-neutral cleavage of DNA. Mechanistic studies indicate that cleavage is initiated by chlorine atoms, produced by charge transfer interaction between chloride anion and AQ triplet states. High-resolution gel electrophoresis suggests that cleavage arises from abstraction of a hydrogen atom from C-4' of deoxyribose units. The targeting of this hydrogen, which is located in the minor groove of duplex DNA, can be effectively blocked by netropsin and, to a lesser degree, berenil, leading to photofootprinting of these minor groove-binding drugs.

AT selectivity and DNA minor groove binding: modelling, NMR and structural studies of the interactions of propamidine and pentamidine with d(CGCGAATTCGCG) 2

A molecular modelling strategy has been developed to identify potential binding sites for bis(amidine) ligands in the minor groove of duplex DNA. Calculations of interaction energy for propamidine and pentamidine with d(CGCGAATTCGCG) 2 show that this duplex contains two symmetrically equivalent binding sites of identical affinity, each displaced by 0.3–0.4 bp from the centre of the AT segment. The ligands occupy groove sites spanning ∼4 and 4–5 bp, respectively, with asymmetric binding to the 5′-AATT sequence. The DNA-bis(amidine) interactions have been examined by high-resolution 1H-NMR. The patterns of induced changes in DNA proton chemical shift and the DNA-ligand NOEs confirm that both agents bind in the AT minor groove in a non-centrosymmetric fashion. Detailed structures were determined for each complex using a NOE-restrained simulated annealing procedure, showing that the B-type DNA conformation is not significantly altered upon complexation with either ligand. The free DNA duplex has previously been shown to be extensively hydrated in the minor groove [Kubinec, M.G. and Wemmer, D.E. (1992) J. Am. Chem. Soc. 114, 8739–8740; Liepinsh, E., Otting, G. and Wüthrich, K. (1992) Nucleic Acids Res. 20, 6549–6553]. We detect hydration water close to the A(H2) protons in the presence of propamidine, which may stabilise certain waters against exchange. This conclusion supports recent crystallographic analyses, suggesting that such ligands may use water molecules to bridge between amidinium protons and host DNA bases. Details of the ligand interactions with AT-tract DNA duplexes can now be compared for the subsequences 5′-AAT, 5′-AATT and 5′-AAATTT.

DNA–Carbohydrate Recognition: Design and Synthesis of an Eight‐Base Sequence‐Selective DNA‐Binding Oligosaccharide

A decacyclic oligosaccharide was designed to dock into the minor groove of duplex DNA selectively at a TCCTXXAGGA sequence. Competition experiments with calicheamicin γ revealed that the oligosaccharide inhibits DNA cleavage and exhibits high DNA binding affinity.

ChemInform Abstract: Synthesis of Radiosensitizers Designed to Bind to the Minor Groove of Duplex DNA.

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The synthesis of radiosensitizers designed to bind to the minor groove of duplex DNA

The synthesis is described of novel compounds, 17, 21, 25 and 27, containing in their molecular architecture both a terminal nitroarene component and an oligopeptide similar to that in the antibiotic distamycin, which is known to bind into the minor groove of duplex DNA. It is hoped that the compounds may concentrate around DNA in cells and so potentiate the action of the nitroarene as a radiosensitizer.