Features of distamycin preferential binding sites on natural DNA predicted using differential scanning calorimetry.

The interaction of distamycin with ColE1 DNA was examined by using differential scanning calorimetry (DSC) taking the helix-coil transition theory of DNA into consideration. Our results here strongly indicate that the affinity of distamycin to DNA, at a low distamycin concentration, depends highly on the DNA sequence, and preferential binding occurs to the sites of four to six successive A-T pairs having two or more successive G-C pairs on both their ends.     

Characterization of distamycin A binding to damaged DNA

We previously reported that distamycin A, a natural antibiotic known as a minor groove binder, could bind to DNA duplexes containing the (6-4) photoproduct formed at its target site, whereas the binding was not observed for duplexes containing the cis-syn cyclobutane pyrimidine dimer in the same sequence context. In this study, we have further analyzed the binding of this drug to lesion-containing duplexes to elucidate its damaged-DNA recognition mechanism. Surface plasmon resonance measurements using various types of DNA showed that distamycin A could bind to several types of lesion-containing DNA. Curve fitting of the CD titration data revealed that the complex formation occurred with K(d) values around 10(-6) and a stoichiometry of 1:1. The results obtained in this study suggested that distamycin A binds to damaged DNA in the same way as to the normal target site, by recognizing the chemical structure of the minor groove.     

Selective binding of actinomycin D and distamycin A to DNA.

The exact sites at which a number of drugs inhibit the nick translation of DNA by E.coli DNA polymerase-I have been pinpointed. In order to do this, a method has been developed for sequencing double-stranded plasmid DNA from the site of a specifically induced nick. The initial experiments have concentrated on analysis of drug inhibition of nick translation in a 200 nucleotide region near the Eco Rl origin of pBR313. Many drugs were found to inhibit nick translation in a highly sequence specific manner. For actinomycin D, significant inhibition occurred at just four sites in the nucleotide sequence under test and only one sequence (pGpCpGpCpGpGp) gave really strong inhibition. Distamycin A gave a different pattern of inhibition with particularly strong stops in just two of the many A-T rich regions in the DNA. Experiments with caffeine suggest that factors in addition to primary sequence are important in determining where major inhibition occurs.     

Targeting DNA quadruplexes with distamycin A and its derivatives: an ITC and NMR study

The use of small molecules that bind and stabilize G-quadruplex structures is emerging as a promising way to inhibit telomerase activity in tumor cells. In this paper, isothermal titration calorimetry (ITC) and (1)H NMR studies have been conducted to examine the binding of distamycin A and its two carbamoyl derivatives (compounds 1 and 2) to the target [d(TGGGGT)](4) and d[AG(3)(T(2)AG(3))(3)] quadruplexes from the Tetrahymena and human telomeres, respectively. The interactions were examined using two different buffered solutions containing either K(+) or Na(+) at a fixed ionic strength, to evaluate any influence of the ions present in solution on the binding behaviour. Experiments reveal that distamycin A and compound 1 bind the investigated quadruplexes in both solution conditions; conversely, compound 2 appears to have a poor affinity in any case. Moreover, these studies indicate that the presence of different cations in solution affects the stoichiometry and thermodynamics of the interactions.     

Synthesis and DNA binding properties of saturated distamycin analogues

A series of saturated heterocyclic analogues of distamycin were prepared and examined. A fluorescent intercalator displacement (FID) assay conducted on p[dA]-p[dT] DNA to obtain C(50) values and a hairpin deoxyoligonucleotide containing an A/T-rich binding site was used to evaluate DNA binding affinity. It is observed that saturated heterocycles greatly reduce the DNA binding relative to distamycin.     

Cytotoxic halogenoacrylic derivatives of distamycin A

The design, synthesis, in vitro and in vivo activities of a series of halogenoacrylic derivatives of distamycin A are described. The structure-activity relationships indicate a key role of the reactivity of alpha-halogenoacrylic moiety. The reactivity and the putative alkylating mechanism of these compounds are different from those of the nitrogen mustards and possibly based on a Michael type reaction. This supports the hypothesis that these compounds represent a class of minor groove binders mechanistically different from tallimustine.     

Quantum yields and fluorescence lifetimes of acridine derivatives interacting with DNA

Interactions of several acridine dyes with DNA from different species were studied by measuring fluorescence lifetimes in the 2–30-nsec range, using the single-photon counting technique, and by measuring fluorescence quantum yields in the steady state. The results confirm the existence of two principal site classes, one in which the dye fluorescence is quenched by interaction with guanine and another in which fluorescence results from the hydrophobic environment of the A·T base pairs. The emitting sites are found, in some cases, to exhibit fluorescent decay curves which can be resolved into two exponential components corresponding to a short and to a long lifetime. The deviation from one exponential component is particularly clear with rivanol, 9-aminoacridine, and quinacrine, with which one component is two or three times longer than the other. The relative proportion of these two components depends only slightly on the DNA base composition and does not depend on the nature of the acridine derivatives. We postulate that this lifetime heterogeneity corresponds to the two discrete steps in the complex formation elucidated by kinetic studies: the first step corresponds to a semi-intercalated, or “external,” dye with a short fluorescence lifetime and the second step corresponds to a totally intercalated dye with a long lifetime. In this model, we assumed that a transient opening of the site near a semi-intercalated dye induces solvent diffusion which in turn is responsible for its short-lived fluorescence.     

Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers

The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at antibiotic/nucleotide ratios up to at least 0.05. CD spectra obtained using DNA model polymers reveal that netropsin binds tightly to poly (dA) · poly (dT), poly (dA-dT) · poly(dA-dT) and poly (dI-dC) · poly (dI-dC) but poorly, if at all, to poly (dG) · poly (dC). Binding curves obtained with calf thymus DNA reveal one netropsin-binding site per 6.0 nucleotides (K_a=2.9 · 10⁵ M⁻¹); corresponding values for distamycin A are one site per 6.1 nucleotides with K_a= 11.6 · 10⁵ M⁻¹. Binding sites apparently involve predominantly A·T-rich sequences whose specific conformation determines their high affinity for the two antibiotics. It is suggested that the binding is stabilized primarily by hydrogen bonding and electrostatic interactions probably in the narrow groove of the DNA helix, but without intercalation. Any local structural deformation of the helix does not involve unwinding greater than approximately 3° per bound antibiotic molecule.     

Synthesis and DNA binding properties of an amidine-linked and phenyl-containing analogue of distamycin A

The synthesis of a novel amidine-linked analogue 1 of the phenyl-containing congener 2 of distamycin A and its DNA binding properties are described. The amidine group in 1 improves its water solubility while retaining the minor groove and AT sequence binding selectively.A phenyl-containing and amidine-linked analogue 1 of distamycin A has improved water solubility while retaining the minor groove and AT sequence binding selectivity to DNA.     

Phenyl sulfur mustard derivatives of distamycin A

The design, synthesis, and cytotoxic activity of novel benzoyl and cinnamoyl sulfur mustard derivatives of distamycin A are described and structure activity relationships are discussed. These sulfur mustards are more potent cytotoxics than corresponding nitrogen mustards in spite of the lower alkylating power, while their sulfoxide analogues are substantially inactive. Cinnamoyl sulfur mustard derivative (7) proved to be one of the most active distamycin-derived cytotoxics, about 1000 times more potent than melphalan.     

Configurational entropy change of netropsin and distamycin upon DNA minor-groove binding

Binding of a small molecule to a macromolecular target reduces its conformational freedom, resulting in a negative entropy change that opposes the binding. The goal of this study is to estimate the configurational entropy change of two minor-groove-binding ligands, netropsin and distamycin, upon binding to the DNA duplex d(CGCGAAAAACGCG).d(CGCGTTTTTCGCG). Configurational entropy upper bounds based on 10-ns molecular dynamics simulations of netropsin and distamycin in solution and in complex with DNA in solution were estimated using the covariance matrix of atom-positional fluctuations. The results suggest that netropsin and distamycin lose a significant amount of configurational entropy upon binding to the DNA minor groove. The estimated changes in configurational entropy for netropsin and distamycin are -127 J K(-1) mol(-1) and -104 J K(-1) mol(-1), respectively. Estimates of the configurational entropy contributions of parts of the ligands are presented, showing that the loss of configurational entropy is comparatively more pronounced for the flexible tails than for the relatively rigid central body.     

Comparison of binding sites in DNA for berenil, netropsin and distamycin. A footprinting study

Techniques of DNase I and micrococcal nuclease footprinting have been used to compare the binding sites for berenil, netropsin and distamycin on two different DNA fragments. Each ligand binds to the A + T-rich zones which contain clusters of at least four A.T base pairs. Neither guanosine nor cytidine nucleotides appear to be allowed within the A + T-rich runs which constitute the preferred binding sites, although they are sometimes protected from DNase I cleavage in neighbouring regions. Berenil and netropsin share with distamycin the property of causing enhanced rates of cleavage at certain sequences flanking their binding sites. There are significant differences in the concentrations of each ligand required to produce defined patterns of protection, seemingly dependent upon the nature (and possibly the gross base composition) of the piece of DNA being used in the experiment.     

Fluorescence-determined preferential binding of quinacrine to DNA.

Quinacrine complexes with native DNA (Calf thymus, Micrococcus lysodeikticus, Escherichia coli, Bacillus subtilis, and Colstridium perfringens) and synthetic polynucleotides (poly(dA) . poly(dT), poly[d(A-T)] . poly[d(A-T)], poly(dG) . poly(dC) and poly[d(G-C)] . poly[d(G-C)]) has been investigated in solution at 0.1 M NaCl, 0.05 M Tris HCl, 0.001 M EDTA, pH 7.5, at 20 degrees C. Fluorescence excitation spectra of complexes with dye concentration D = 5-30 microM and DNA phosphate concentration P = 400 microM have been examined from 300 to 500 nm, while collecting the emission above 520 nm. The amounts of free and bound quinacrine in the dye-DNA complexes have been determined by means of equilibrium dialysis experiments. Different affinities have been found for the various DNAs and their values have been examined with a model that assumes that the binding constants associated with alternating purine and pyrimidine sequences are larger than those relative to nonalternating ones. Among the alternating nearest neighbor base sequences, the Pyr(3'-5')Pur sequences, i.e., C-G, T-G, C-A and T-A seem to bind quinacrine stronger than the remaining sequences. In particular the three sites, where a G . C base pair is involved, are found to display higher affinities. Good agreement is found with recent calculations on the energetics of intercalation sites in DNA. The analysis of the equilibrium shows also that the strength of the excitation spectrum of bound dye depends strongly upon the ratio of bound quinacrine to DNA. This effect can be attributed to dye-dye energy transfer along DNA.     

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.     

Selection of DNA nanoparticles with preferential binding to aggregated protein target

High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, life''s own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions.     

Nuclear protein factors binding with specific DNA sequences

Primary structure of thousands of genes is being determined in many laboratories worldwide. While it is relatively easy to analyse the coding region(s) of genes, it is usually hard to understand what is located in non-coding regions. A non-coding region may contain very valuable information about the mode of functioning of a given gene, e. g. promoters, enhancers, silencers etc. The regulatory function of these sequences is determined by their interaction with certain sequence-specific proteins, i. e. the presence of a certain DNA sequence in a non-coding region of a gene may suggest that the gene is regulated by a specific protein factor. This minireview summarizes recent data on most known eukaryotic sequence-specific DNA-binding protein factors, including their origin, DNA consensus, and their role in expression of corresponding genes.     

Hydroxyl radical footprinting of the sequence-selective binding of netropsin and distamycin to DNA

Hydroxyl radicals, generated by allowing an iron (II).EDTA complex to react with hydrogen peroxide, have been employed to cleave the 160-base pair tyrT DNA fragment in the presence and absence of the minor groove-binding antibiotics netropsin and distamycin A. The control DNA cleavage pattern is practically independent of nucleotide sequence, which overcomes certain limitations of other footprinting techniques, so that additional information can be gained about the AT-rich sequence preference of the minor groove-binding ligands.     

DNA polyintercalation: comparison of DNA binding properties of an acridine dimer and trimer

The DNA binding characteristics of a mono-, di- and trimeric derivative of 9-aminoacridine were studied. The length of the linking carboxamidoalkyl chains was selected to allow bis- or tris-intercalation according to the excluded-site model. Measurements of DNA unwinding angle using closed circular DNA showed that the trimeric derivative behaves as a tris-intercalating agent. Nevertheless the increase of DNA binding affinity on going from dimer to trimer was found to be relatively small. This is probably related to the large structural constraint for DNA binding of the trimeric derivative. The nature of the linking chain for the design of high-affinity DNA poly-intercalating agents appears therefore critical.     

Interactions between distamycin A analogs bound to DNA

The experimental binding isotherms of the distamycin A analog to 8 natural and synthetic DNAs were analyzed. The shapes of binding isotherms suggest that the bound ligand molecule induces transitions of DNA (B-form) into two perturbated conformation states. These transitions are responsible for the existence of positive and negative cooperative effects on binding of distamycin analogs to DNA. At low levels of binding positive cooperative effects play a dominating role whereas at high levels of binding negative cooperative effects are observed. These cooperative effects can be described by the aid of a potential of pairwise interactions between nearest neighbour bound antibiotic molecules. A detailed analysis of experimental binding isotherms shows that characteristic distances over which these interactions are extended depend on the AT content of DNA. The energetical and structural parameters characterising the allosteric transitions of DNA to the perturbated states are obtained.