Noncovalent DNA binding of bis(1,10-phenanthroline)copper(I) and related compounds

The noncovalent DNA binding of the bis(1,10-phenanthroline)copper(I) complex [(Phen)2CuI] was examined under anaerobic conditions by absorption and circular dichroism spectroscopy, and viscometry, as a function of phenanthroline concentration. Analyses according to the McGhee-von Hippel method indicated that binding exhibited both neighbor-exclusion and positive cooperativity effects, with a neighbor-exclusion parameter n approximately 2 and a cooperativity parameter omega approximately 4. The association constant for (Phen)2CuI binding decreased with increasing concentration of phenanthroline in excess over that required to stoichiometrically generate (Phen)2CuI, indicating that free phenanthroline was a weak competitive inhibitor of (Phen)2CuI binding. The maximal association constant for DNA binding of (Phen)2CuI in 0.2 M NaCl and 9.8% ethanol, extrapolated to zero concentration of excess phenanthroline, was 4.7 x 10(4) M-1 (DNA base pairs). The magnitude of the neighbor-exclusion parameter, the changes in spectral properties of (Phen)2CuI induced by DNA binding, and the increase in DNA solution viscosity upon (Phen)2CuI addition are consistent with a model for DNA binding by (Phen)2CuI involving partial intercalation of one phenanthroline ring of the complex between DNA base pairs in the minor groove as suggested previously [Veal & Rill (1989) Biochemistry 28, 3243-3250]. Viscosity measurements indicated that the mono(phenanthroline)copper(I) complex also binds to DNA by intercalation; however, no spectroscopic or viscometric evidence was found for DNA binding of free phenanthroline or the bis(2,9-dimethyl-1,10-phenanthroline)copper(I) complex. DNA binding of free phenanthroline may be cooperative and induced by prior binding of (Phen)2CuI.     

Interaction of a macrocyclic bisacridine with DNA

The binding of the macrocycle SDM to DNA was investigated by visible spectroscopy, stopped-flow kinetics, and NMR spectroscopy. SDM is composed of two 9-aminoacridines linked via the amino groups by a spermine side chain and via the 4-positions by a N,N'-[(methylthio)ethyl]succinamide side chain [Zimmerman, S. C., Lamberson, C. R., Cory, M., & Fairley, T. A. (1989) J. Am. Chem. Soc. 111, 6805-6809]. The visible spectrum of SDM bound to poly[d(A-T)]2 or poly[d(G-C)]2 is red-shifted relative to the spectrum of SDM alone and displays considerable hypochromicity. Results from titrations of SDM with polymer indicate a binding site size of three base pairs per macrocycle. The dissociation constant for SDM bound to either poly[d(A-T)]2 or poly[d(G-C)]2 is an order of magnitude lower than that for a similar bisacridine linked only by a spermine side chain. In addition, the dependence of the dissociation constant on ionic strength is significantly reduced. NMR studies of SDM complexes with poly[d(A-T)]2 or a tetramer, d(CGCG)2, show that intercalation is the mode of binding. The magnitudes of the chemical shift differences for SDM aromatic protons in the free and bound states support intercalation with the acridine ring systems essentially parallel to the long axis of the base pairs. Cross peaks from NOESY spectra of the SDM complex with d(CGCG)2 further support this mode of binding and provide information on the structure of the complex. The results are analyzed for consistency with each of three binding models: (i) bisintercalation with the two side chains in the same groove; (ii) bisintercalation according to the neighbor-exclusion principle with the two side chains in opposite grooves; and (iii) bisintercalation with two side chains in opposite grooves but with violation of the neighbor-exclusion principle. Model i is found to be unlikely on the basis of all evidence obtained, including preliminary modeling studies. Both models ii and iii can be reconciled with the experimental evidence and from a modeling standpoint are energetically feasible.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

Binding of ligands to a one-dimensional heterogeneous lattice. II. Intercalation of tilorone with DNA and polynucleotides

The interaction of tilorone with DNA and five synthetic polydeoxyribonucleotides [(I): poly[d(A-T)]·poly[d(A-T)]; (II): poly[d(A-C)]·poly[d(G-T)]; (III): poly[d(G-C)]·poly[d(G-C)]; (IV): poly(dG)·poly(dC); and (V): poly(dA)·poly(dT)] has been investigated. Binding isotherms for the homopolymers were obtained by microdialysis equilibria using ¹⁴C-labeled tilorone and interpreted with different models: exclusion effect, associated or not associated with cooperativity, or variable exclusion. Affinity appears to be related more to local structure than to base composition and decreases in the following order: (I) > (II) > (III) > (IV) > (V). Intercalation in circular DNA was demonstrated by electrophoresis migration and electron microscopy, which yielded an average unwinding angle of 7° per bound dye. The behavior observed in CD and UV spectroscopy shows a sequence similar to the affinities. Tilorone seems to be less intercalated in (IV) and not at all in (V). The experimental binding isotherm of tilorone to DNA was well fitted on the basis of a model where DNA acts as a heterogeneous lattice built with the six different possible couples of adjacent base pairs, each potential site behaving as if it were in the corresponding homopolymer. The results are discussed in terms of specificity of alternating Pyr-Pur sequences and related to theoretical calculations on intercalation energies of DNA.     

DNA Intercalation and Photoinduced Cleavage by 4-Nitro(N-Hexylamine)1,8-Naphthalimide

Abstract

A novel intercalator, 4-nitro(N-hexylamine)1,8-naphthalimide, was synthesised and its DNA binding and photoinduced DNA cleavage properties were studied. The DNA unwinding results show that it binds through intercalation. Absorption and fluorescence spectroscopy reveal the preference for A/T base pairs as compared to G/C base pairs for the binding. The intercalator produces photoinduced single strand scissions in double helical DNA.     

Conformations of duplex structures formed by oligodeoxynucleotides covalently linked to the intercalator 2-methoxy-6-chloro-9-aminoacridine

A family of covalent complexes between oligonucleotides and derivatives of the intercalating agent 9-amino acridine has been synthesized (Asseline, U., Thuong, N.T. and Helene, C. (1983) C.R.Acad. Sci. (Paris) 297 (III), 369-372) and studied (Lancelot, G., Asseline, U., Thuong, N.T., and Helene, C. (1985) Biochemistry 24, 2521-2529; Lancelot, G., Asseline, U., Thuong, N.T., and Helene, C. (1985) J. Biomol. Str. Dyn. 3, 913-921) with a view to understand nucleic acid-nucleic acid recognition. In order to understand the nature of interactions between the intercalator and the oligonucleotides in such complexes and the sensitivity of such interactions to the polymorphic form of the DNA, we have carried out molecular mechanics simulations on duplex deoxyoligonucleotides d(A)6.d(T)6 (A and B forms) and d(TATC).d(GATA) (B form) covalently bound to 2-methoxy-6-chloro-9-aminoacridine through a pentamethylene linker chain. Structures in which the acridine derivative is end stacked (at the 3' and 5' ends) and in which the dye is intercalated between the terminal base pairs (at both the ends) and between second and third base pairs from the 3' end are all of reasonably low energy in both A and B forms of DNA. Our studies on 3' end complexes find that in the B form, intercalation of the dye between the second and third base pairs is preferred over the other two modes of binding, while in the A form, intercalation between the terminal base pairs is preferred. In the 5' end A and B form complexes, outside stacking and intercalation between the terminal base pairs are preferred, respectively. Our calculations suggest the possibility that the presence of the dye attached covalently to the DNA can induce conformational transitions in the DNA. For example, intercalation of the dye two base pairs from the end could induce an A----B transition.     

Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif

We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.     

Anticooperative Binding Governs the Mechanics of Ethidium-Complexed DNA

DNA intercalators bind nucleic acids by stacking between adjacent basepairs. This causes a considerable elongation of the DNA backbone as well as untwisting of the double helix. In the past few years, single-molecule mechanical experiments have become a common tool to characterize these deformations and to quantify important parameters of the intercalation process. Parameter extraction typically relies on the neighbor-exclusion model, in which a bound intercalator prevents intercalation into adjacent sites. Here, we challenge the neighbor-exclusion model by carefully quantifying and modeling the force-extension and twisting behavior of single ethidium-complexed DNA molecules. We show that only an anticooperative ethidium binding that allows for a disfavored but nonetheless possible intercalation into nearest-neighbor sites can consistently describe the mechanical behavior of intercalator-bound DNA. At high ethidium concentrations and elevated mechanical stress, this causes an almost complete occupation of nearest-neighbor sites and almost a doubling of the DNA contour length. We furthermore show that intercalation into nearest-neighbor sites needs to be considered when estimating intercalator parameters from zero-stress elongation and twisting data. We think that the proposed anticooperative binding mechanism may also be applicable to other intercalating molecules.     

DNA bis-intercalation: Theoretical analysis,including cooperativity of the interaction of echinomycin analogs with DNA

A theoretical analysis of the binding curves for the bifunctional peptide antibiotic echinomycin and its analogs interacting with a variety of DNAs is presented. The method is an extension of our previous work using the sequence-generating-function technique and has been modified to allow for consideration of cooperativity within the framework of the neighbor-exclusion rule. Binding by both single and double intercalation is included, and the results are, in many cases, superior to the analysis based on single-mode binding. In all cases the data are consistent with the neighbor-exclusion principle for intercalation when analyzed with this approach, while data analysis, using a simpler method, often led to violation of this rule. Positive cooperativity is considered in the treatment of data for binding to poly(dA-dT). Finally, we discuss the relationship between base specificity and binding site size.     

121 Influence of changes in DNA conformation on thermodynamic contribution during interaction of human genomic DNA and its mutant with anticancer drugs

Isothermal calorimetry (ITC) is efficient in characterizing and recognizing both high affinity and low affinity intermolecular interactions quickly and accurately. Adriamycin (ADR) and daunomycin (DNM) are the two anticancer drugs whose activity is achieved mainly by intercalation with DNA. During chemotherapy, normal human genomic DNA and mutated DNA from K562 leukemic cells show different thermodynamic properties and binding affinities on interaction with ADR and DNM when followed by ITC. Normal DNA shows more than one step in kinetic analysis, which could be attributed to outside binding, intercalation and reshuffling as suggested by Chaires et al. (1985); whereas K562 DNA fits a different binding pattern with higher binding affinities (by one order or more) compared to normal DNA. Structural properties of the interaction were followed by laser Raman spectroscopy, where difference in structure was apparent from the shifts in marker B DNA Raman bands (Ling et al., 2005). A correlation of thermodynamic contribution and structural data reveals step wise changes in normal genomic DNA conformation on drug binding. The overall structural change is higher in normal DNA–DNM interaction suggesting a partial B to A transition on drug binding. Such large changes were not observed for K562 DNA–DNM interaction which showed B to A transition properties in native from itself corroborating with our earlier findings (Ghosh et al., 2012).     

抗菌肽BuforinⅡ衍生物与大肠杆菌基因组DNA的作用机制

【目的】研究抗菌肽BuforinⅡ的衍生物BF2-A/B与大肠杆菌基因组DNA的作用机制。【方法】琼脂糖电泳检测肽对DNA的断裂作用,凝胶阻滞实验研究肽与DNA的结合作用,圆二色谱考察结合肽后DNA结构的变化,荧光光谱分析肽与溴化乙锭竞争性嵌入DNA以及磷酸根对肽与DNA相互作用的影响。【结果】BF2-A/B不断裂基因组DNA而是结合DNA,使DNA双螺旋结构变得松散,削弱碱基对间的堆积作用,并取代EB,使EB-DNA复合体系荧光减弱。而PO43-的加入减弱了肽对DNA-EB荧光的淬灭作用。【结论】衍生肽与DNA的结合方式是先靠静电引力吸附到DNA磷酸基团上,随即插入双螺旋沟槽,嵌入碱基对间。BF2-B有更多的正电荷,更强的插入沟槽和嵌入碱基对的能力,使得其结合DNA的能力比BF2-A强。     

Spectroscopic and calorimetric studies on the DNA recognition of pyrrolo[2,1-c][1,4]benzodiazepine hybrids

DNA binding of two hybrid ligands composed of an alkylating pyrrolo[2,1-c][1,4]benzodiazepine (PBD) moiety tethered to either a naphthalimide or a phenyl benzimidazole chromophore was studied by DNA melting experiments, UV and fluorescence titrations, CD spectroscopy and isothermal titration calorimetry (ITC). Binding of both hybrids results in a remarkable thermal stabilization with an increase of DNA melting temperatures by up to 40 °C for duplexes that allow for a covalent attachment of the PBD moiety to guanine bases in their minor groove. CD spectroscopic measurements suggest that the naphthalimide moiety of the drug interacts through intercalation. In contrast, the PBD-benzimidazole hybrid binds in the DNA minor groove with a preference for (A,T)₄G sequences. Whereas the binding of both ligands is enthalpy-driven and associated with a negative entropy, the benzimidazole hybrid exhibits a less favourable binding enthalpy that is counterbalanced by a more favourable entropic term when compared to the naphthalimide hybrid.     

The presence of two modes of binding to calf thymus DNA by metal-free bleomycin: a low frequency Raman study

Double-stranded DNA is targeted by bleomycin in cancer cells and ambiguity exists as to its mode of DNA binding. A conventional Raman study was performed on drug/DNA complexes in which the low frequency spectral region (560-930 cm(-1)) was examined at two temperatures (19 and 30 degrees C). At 30 degrees C, a global Raman hypochromism was observed consistent with partial intercalation of the bithiazole moiety. At 19 degrees C, Raman hypochromism (increased base pair stacking) was detected for bands associated with GC base pairs while Raman hyperchromism (base pair destacking) was evident for bands associated with AT base pairs. These results suggest that intercalation of the bithiazole moiety occurs with greater disruption of the more efficiently stacked AT base pairs at the lower temperature. Evidence for minor groove binding was indicated by an increase in the population of bands corresponding to C3' endo sugar conformations resulting from drug induced local desolvation of the DNA polymer.     

Analysis of mixed DNA‐bisnaphthalimide interactions involving groove association and intercalation with surface‐based and solution methodologies

The bisnaphthalimide cytotoxic agent elinafide exhibits a mixed DNA binding mode including groove‐association and intercalation. We have compared the interaction of elinafide and two bisnaphthalimide analogues with various natural and modified DNA sequences using solution NMR and UV‐melting methods and surface plasmon resonance (SPR) experiments at different pH conditions. The combined data obtained with these techniques established a high‐affinity binding mode comprising intercalation and strong electrostatic contacts with guanine bases in the major groove, and a weaker interaction with A·T pairs likely involving groove association. However, the SPR binding constants and the NMR and UV‐melting binding parameters responded differently to variations in DNA bases and ligand intercalating moieties. The rates and equilibrium constants determined by SPR clearly responded to changes in pH and DNA groove composition, but were rather insensitive to alterations in drug rings and DNA bases affecting the intercalation process. Conversely, the intermolecular stacking interactions detected by NMR and the ligand‐induced thermal stabilizations measured by UV depended on both sets of factors and were controlled by the sequence‐dependent properties of the DNA helices, indicating that these data were modulated by naphthalimide stacking in addition to groove association. A two‐step binding process where a groove‐bound state is required prior to intercalation is proposed as an explanation for these observations. These findings may be useful for studying other classes of DNA‐ and RNA‐binding drugs, which frequently combine groove‐binding and stacking moieties. © 2012 Wiley Periodicals, Inc. Biopolymers 97:974–987, 2012.     

Unspecific DNA binding of the DNA binding domain of the glucocorticoid receptor studied with flow linear dichroism

The unspecific interaction between the DNA-binding domain of the human glucocorticoid receptor and DNA was studied using linear dichroism (LD) and circular dichroism (CD) spectroscopy. The amplitude of the LD signal was found to increase upon addition of protein at ionic strengths less than 60 nM Na+, indicating an increased persistence length of the complex compared to uncomplexed DNA. Analysis of the LD spectrum suggests that the binding does not involve intercalation of tyrosine residues. Evidence of saturation is found at a binding stoichiometry of approximately 5 DNA base pairs per protein monomer.     

Benzo[c]phenanthrene adducts and nogalamycin inhibit DNA transesterification by vaccinia topoisomerase

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N2-deoxyguanosine and N6-deoxyadenosine positions within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the +6 and +5 base pairs, slowed 4-fold by intercalation between the +5 and +4 base pairs, and virtually abolished by BcPh intercalation between the +4 and +3 base pairs and the +3 and +2 base pairs. Intercalation between the +2 and +1 base pairs by the +2R BcPh dA adduct abolished transesterification, whereas the overlapping +1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the +1 and -1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the -1 and -2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the -2 and -3 base pairs had little effect. The anthracycline drug nogalamycin, a non-covalent intercalator with preference for 5'-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC50 of 0.7 microM. Nogalamycin was most effective when the drug was pre-incubated with DNA and when the cleavage target site was 5'-CCCTT/G instead of 5'-CCCTT/A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.     

Biophysical studies of a ruthenium(II) polypyridyl complex binding to DNA and RNA prove that nucleic acid structure has significant effects on binding behaviors

The interactions of a metal complex [Ru(phen)(2)PMIP](2+) {Ru=ruthenium, phen=1,10-phenanthroline, PMIP=2-(4-methylphenyl)imidazo[4,5-f]1,10-phenanthroline} with yeast tRNA and calf thymus DNA (CT DNA) have been investigated comparatively by UV-vis spectroscopy, fluorescence spectroscopy, viscosity measurements, isothermal titration calorimetry (ITC), as well as equilibrium dialysis and circular dichroism (CD). Spectroscopic studies together with ITC and viscosity measurements indicate that both binding modes of the Ru(II) polypyridyl complex to yeast tRNA and CT DNA are intercalation and yeast tRNA binding of the complex is stronger than CT DNA binding. ITC experiments show that the interaction of the complex with yeast tRNA is driven by a moderately favorable enthalpy decrease in combination with a moderately favorable entropy increase, while the binding of the complex to CT DNA is driven by a large favorable enthalpy decrease with a less favorable entropy increase. The results from equilibrium dialysis and CD suggest that both interactions are enantioselective and the Delta enantiomer of the complex may bind more favorably to both yeast tRNA and CT DNA than the Lambda enantiomer does, and that the complex is a better candidate for an enantioselective binder to yeast tRNA than to CT DNA. Taken together, these results indicate that the structures of nucleic acids have significant effects on the binding behaviors of metal complexes.     

How much pi-stacking do DNA termini seek? Solution structure of a self-complementary DNA hexamer with trimethoxystilbenes capping the terminal base pairs

The exposed terminal base pairs of DNA duplexes are nonclassical binding sites for small molecules. Instead, small molecules usually prefer intercalation or minor groove binding. Here we report the solution structure of the DNA duplex (TMS-TGCGCA)(2), where TMS denotes trimethoxystilbene carboxamides that are 5'-tethered to the DNA. The stilbenes, for which intercalation is conformationally accessible, stack on the terminal T:A base pairs of an undisturbed B-form duplex. Two conformations, differing by the orientation of the stilbene relative to the terminal base pair, are observed, indicating that the flip rate is slow for the pi-stacked aromatic ring system. The trimethoxystilbene is known to greatly increase base pairing fidelity at the terminus. Here we show that it gauges the size of the T:A base pair by embracing the 2'-methylene group of the terminal dA residue of the unmodified terminus with its methoxy "arms", but that it does not engage the entire base pair in pi-stacking. Mismatched base pairs with their altered geometry will not allow for the same embracing interaction. On the basis of the current structure, a trimethoxychrysene carboxamide is proposed as a ligand with increased pi-stacking surface and possible applications as improved fidelity-enhancing element.     

Heterogeneous DNA binding modes of berenil.

Isothermal titration calorimetry (ITC) profiles of berenil bound to different DNAs show that, despite the strong preference of berenil for AT-rich regions in DNA, it can bind to other DNA sequences significantly. The ITC results were used to quantify the binding of berenil, and the thermodynamic profiles were obtained using natural DNAs as well as synthetic polynucleotides. ITC binding isotherms cannot be simply described when a single set of identical binding sites is considered, except for poly[d(A-T)2]. Ultraviolet melting of DNA and differential scanning calorimetry were also used to quantify several aspects of the binding of berenil to salmon testes DNA. We present evidence for secondary binding sites for berenil in DNA, corresponding to G+C rich sites. Berenil binding to poly[d(G-C)2] is also observed. Circular dichroism experiments showed that binding to GC-rich sites involves drug intercalation. Using a molecular modeling approach we demonstrate that intercalation of berenil into CpG steps is sterically feasible.