echinomycin and 2-6-diaminopurine

Atomic force microscopy (AFM) has been used to examine the conformational effects of echinomycin, a DNA bis-intercalating antibiotic, on linear and circular DNA. Four different 398 bp DNA fragments were synthesized, comprising a combination of normal and/or modified bases including 2,6-diaminopurine and inosine (which are the corresponding analogues of adenine and guanosine in which the 2-amino group that is crucial for echinomycin binding has been added or removed, respectively). Analysis of AFM images provided contour lengths, which were used as a direct measure of bis-intercalation. About 66 echinomycin molecules are able to bind to each fragment, corresponding to a site size of six base-pairs. The presence of base-modified nucleotides affects DNA conformation, as determined by the helical rise per base-pair. At the same time, the values obtained for the dissociation constant correlate with the types of preferred binding site available among the different DNA fragments; echinomycin binds to TpD sites much more tightly than to CpG sites. The structural perturbations induced when echinomycin binds to closed circular duplex pBR322 DNA were also investigated and a method for quantification of the structural changes is presented. In the presence of increasing echinomycin concentration, the plasmid can be seen to proceed through a series of transitions in which its supercoiling decreases, relaxes, and then increases.

Topics: 2-Aminopurine; DNA; DNA, Circular; Echinomycin; Hydrogen Bonding; Intercalating Agents; Ligands; Microscopy, Atomic Force; Nucleic Acid Conformation; Plasmids

The luzopeptin antibiotics contain a cyclic decadepsipeptide to which are attached two quinoline chromophores that bisintercalate into DNA. Although they bind DNA less tightly than the structurally related quinoxaline antibiotics echinomycin and triostin A, the molecular basis of their interaction remains unclear. We have used the PCR in conjunction with novel nucleotides to create specifically modified DNA for footprinting experiments. In order to study the influence that removal, addition or relocation of the guanine 2-amino group, which normally identifies G.C base pairs from the minor groove, has on the interaction of luzopeptin antibiotics with DNA. The presence of a purine 2-amino group is not strictly required for binding of luzopeptin to DNA, but the exact location of this group can alter the position of preferred drug binding sites. It is, however, not the sole determinant of nucleotide sequence recognition in luzopeptin-DNA interaction. Nor can the selectivity of luzopeptin be attributed to the quinoline chromophores, suggesting that an analogue mode of DNA recognition may be operative. This is in contrast to the digital readout that seems to predominate with the quinoxaline antibiotics.

Topics: 2-Aminopurine; Antiprotozoal Agents; Base Pairing; Base Sequence; Binding Sites; Deoxyribonuclease I; DNA; DNA Footprinting; DNA Replication; DNA, Bacterial; Echinomycin; Electrophoresis, Polyacrylamide Gel; Hydrogen Bonding; Hydroxyquinolines; Inosine; Intercalating Agents; Molecular Sequence Data; Molecular Structure; Peptides, Cyclic; Polymerase Chain Reaction; Quinolines; Quinoxalines; Structure-Activity Relationship

In order to clarify the role of the purine 2-amino group in the recognition of DNA by small molecules we have examined the binding of actinomycin D and echinomycin to artificial DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine (DAP) in one of the complementary strands. These DNAs, prepared by a method based upon PCR, present various potential sites for antibiotic binding, including several containing only a single purine 2-amino group in different configurations. The results show unambiguously that the presence of two 2-amino groups is mandatory for binding of actinomycin D to double-stranded DNA. In the case of echinomycin only one purine 2-amino group is required for remarkably strong binding to the asymmetric TpDAP.TpA dinucleotide step, but the CpDAP.TpI step (which also contains only a single purine-2 amino group) does not afford a binding site. Evidently, removing a 2-amino group (G-->I substitution) is dominant over adding one (A-->DAP substitution). No sequences containing just a single guanine residue are acceptable. The possibility is raised that replacing guanosine with inosine may do more than remove a group endowed with hydrogen bonding capability and interfere with ligand binding in other ways. The new methodology developed to construct asymmetrically substituted DNA substrates for this work provides a novel strategy that should be generally applicable for studying ligand-DNA interactions, beyond the specific interest in drug binding to DNA, and may help to elucidate how proteins and oligonucleotides recognize their target sites.

Topics: 2-Aminopurine; Base Composition; Binding Sites; Cytosine; Dactinomycin; DNA; DNA Footprinting; DNA Primers; Echinomycin; Hydrogen Bonding; Hypoxanthine; Inosine; Molecular Sequence Data; Nucleic Acid Heteroduplexes; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Thymine

The expedient of preparing homologous DNA samples substituted with I for G, DAP for A, or both, has been used to investigate the role of the purine 2-amino group in determining the preferred binding sites for antibiotics on DNA. The selectivity of echinomycin for CpG steps, of actinomycin for GpC steps, and of netropsin for A + T-rich tracts, is seen to be radically altered in the substituted DNA molecules.

Topics: 2-Aminopurine; Adenine; Anti-Bacterial Agents; Autoradiography; Base Sequence; Dactinomycin; Deoxyribonuclease I; DNA; Echinomycin; Guanine; Inosine; Molecular Sequence Data; Netropsin; Nucleic Acid Conformation; RNA, Transfer, Tyr

The binding of echinomycin to DNA hexamers of the form GpApXpZpTpC, where the central XpZ step can be CpG, TpA, GpC, or ApT, has been studied by molecular modeling and molecular mechanics techniques. Interaction energies have also been calculated for the complexation of echinomycin with sequences containing the preferred central CpG step and different flanking base pairs. Besides, two more sets of sequences incorporating either 2,6-diaminopurine (DAP) or hypoxanthine in place of adenine or guanine, respectively, have been examined. The aim of this work was to evaluate the relative importance of hydrogen-bonding and stacking interactions in the association of echinomycin with DNA and further rationalize the experimental evidence. The results of these calculations are in consonance with available data from footprinting experiments and appear to support our previous hypothesis that, in addition to the crucial intermolecular hydrogen bonds in the central region, the stacking interactions involving the quinoxaline-2-carboxamide chromophores of the drug and the DNA base pairs play an important role in modulating the binding specificity of this bisintercalating antitumor antibiotic. This is most clearly seen when sequences with similar minor-groove environments are compared (e.g. CpI vs TpA or CpG vs TpDAP). The dipole moment of N-methylquinoxaline-2-carboxamide has been measured (mu = 4.15 +/- 0.03 D) and compares very well with the calculated value (mu = 4.14 D). The fact that G:C, I:C, A:T, and DAP:T base pairs are shown to be endowed with distinct van der Waals and electrostatic stacking properties with respect to this heteroaromatic ring system could have important implications for the design of novel DNA mono- and bis-intercalating agents.

Topics: 2-Aminopurine; Base Composition; Base Sequence; Binding Sites; Chemical Phenomena; Chemistry, Physical; Deoxyribonuclease I; DNA; Echinomycin; Electrochemistry; Hydrogen Bonding; Hypoxanthine; Hypoxanthines; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Quinoxalines; Thermodynamics