Circular dichroism of adenine and thymine containing synthetic polynucleotides

Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A-T)]·poly[d(T-A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double-stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A-t)]·poly[d(T-A)] are due to a change in conformation in which the secondary structure goes from a C- to B-type spectrum by increasing the A-type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B-type geometries occurs.     

Binding of nonintercalative antitumor drugs to DNA-polymers: structural effects of bisquaternary ammonium heterocycles

The binding of the antitumor agents SN-16814 nd SN-13232 to various DNA's in solution was monitored by CD and UV absorption measurements. In addition comparative studies with dA.dT containing duplex DNA of the related ligands SN-6136 and SN-6324 were included with respect to effects of structural variations. In general all four ligands show a dA.dT preference in their binding affinity to DNA. Differences were observed for the reaction of SN-16814 which contains bicyclic ring system: it has a lower base pair selectivity, shows some affinity to poly(dG-dC).poly(dG-dC), poly(rA).poly(rU) and poly(rU). The binding mechanism of SN-16814 is associated with a significant time dependent binding effect in CD spectra and UV absorption in case of reaction with poly(dA).poly(dT) and poly(dI).poly(dC) indicating a slow kinetics. The preferred binding to dA.dT base pairs in DNA decreases in the order from SN-61367 greater than SN-13232 greater than SN-6324,SN-16814 as judged from CD titration studies, salt dissociation and melting temperature data. Competitive binding experiments with netropsin (Nt) or distamycin-5 revealed that SN-16814 and SN-13232 are displaced from poly(dA.dT).poly(dA-dT) suggesting that both ligands are less strongly bound than Nt and Dst-5 within the minor groove of B-DNA. These studies are consistent with results of the DNAse I cleavage of poly(dA-dT).poly(dA-dT) which show the same relative order of inhibition of the cleavage reaction due to ligand binding. The results suggest that the variability of the DNA binding and dA.dT sequence specificity may reside in the adaptability of benzamide-type ligands in the helical groove which is influenced by distinct structural modifications of the ligand conformation.     

Binding of Nonintercalative Antitumor Drugs to DNA-Polymers: Structural Effects of Bisquaternary Ammonium Heterocycles

Abstract

The binding of the antitumor agents SN-16814 nd SN-13232 to various DNA's in solution was monitored by CD and UV absorption measurements. In addition comparative studies with dA · dT containing duplex DNA of the related ligands SN-6136 and SN-6324 were included with respect to effects of structural variations. In general all four ligands show a dA · dT preference in their binding affinity to DNA.

Differences were observed for the reaction of SN-16814 which contains bicyclic ring system: it has a lower base pair selectivity, shows some affinity to poly(dG-dC) · poly(dG-dC), poly(rA) · poly(rU) and poly(rU). The binding mechanism of SN-16814 is associated with a significant time dependent binding effect in CD spectra and UV absorption in case of reaction with poly(dA) · poly(dT) and poly(dI) · poly(dC) indicating a slow kinetics.

The preferred binding to dA · dT base pairs in DNA decreases in the order from SN-61367 > SN-13232 > SN-6324, SN-16814 as judged from CD titration studies, salt dissociation and melting temperature data. Competitive binding experiments with netropsin (Nt) or distamycin-5 revealed that SN-16814 and SN-13232 are displaced from poly(dA-dT) · poly(dA-dT) suggesting that both ligands are less strongly bound than Nt and Dst-5 within the minor groove of B-DNA. These studies are consistent with results of the DNAase I cleavage of poly(dA-dT) · poly(dA-dT) which show the same relative order of inhibition of the cleavage reaction due to ligand binding. The results suggest that the variability of the DNAbinding and dA · dT sequence specificity may reside in the adaptability of benzamide-type ligands in the helical groove which is influenced by distinct structural modifications of the ligand conformation.     

Investigation of DNA binding modes for a symmetrical cyanine dye trication: effect of DNA sequence and structure.

The DNA binding behavior of a tricationic cyanine dye (DiSC3+(5)) was studied using the [Poly(dA-dT)]2, [Poly(dI-dC)]2 and Poly(dA) x Poly(dT) duplex sequences and the Poly(dA) x 2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA) x Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]2 duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]2 sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J.L., et al. (1999) J. Am. Chem. Soc. 121, 2987-2995] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.     

The binding of CC-1065 to thymidine and deoxyadenosine oligonucleotides and to poly(dA).poly(dT)

In this work, we report on the binding of the novel antitumor agent CC-1065 to poly(dA).poly(dT) and to mixtures of dA and dT oligomers as determined by electronic absorption and circular dichroism (CD) methods. In addition, the DNA binding properties of CC-1065 and its binding mechanism are compared to those of netropsin. CC-1065 binds to the polymer by at least three mechanisms to produce one irreversibly and two reversibly bound species. One reversibly bound species is moderately stable, but in time (days), it converts to the irreversibly bound species. Both of these species bind within the minor groove of the polymer and exhibit intense CC-1065 induced CD spectra. The other reversibly bound species does not acquire an induced CD. CC-1065 forces B-form duplex formation between mixtures of single strand dA and dT oligomers and binds irreversibly to the duplexes without showing the presence of an intermediate, reversibly bound species. The induced CD increases with increasing length of the oligomer, from the 5-mer (barely detectable CD) to the 14-mer (intense CD). The 7-, 10- and 14-mer mixtures bind about 1, between 1 and 2, and between 2 and 3 CC-1065 molecules, respectively. Computer graphic models of the CC-1065-DNA complex show that the covalent adduct of CC-1065 and unreacted CC-1065 can attain the same close van der Waals contacts between adenine C2 hydrogens and antibiotic CH groups that were observed in the crystal structure of the netropsin-DNA complex. These contacts may account for the dA-dT base pair binding specificity of CC-1065 and for the stability of the reversibly bound CC-1065 species.     

Spectroscopic analysis of the interaction of Escherichia coli DNA-dependent RNA polymerase with T7 DNA and synthetic polynucleotides.

We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA.     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA)·poly(dT) and poly(dG)·poly(dC), and with triple helical poly(dA)·[poly(dT)]₂ and poly(dC)·poly(dG)·poly(dC)⁺ were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA)·poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG)·poly(dC) and -poly(dC)·poly(dG)·poly(dC)⁺ complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

Infrared and Raman studies show that poly(dA).poly(dT) and d(AAAAATTTTT)2 exhibit a heteronomous conformation in films at 75% relative humidity and a B-type conformation at high humidities and in solution

The decadeoxynucleotide d(AAAAATTTTT)2 in duplex form and the double-helical polynucleotide poly(dA).poly(dT) have been studied by Raman and infrared (IR) spectroscopy under a variety of environmental conditions. The IR spectra have been taken of cast films and compared to the IR spectra of the alternating poly(dA-dT), which shows clear B-genus and A-genus vibrational spectra under conditions of high (greater than 92%) and low (75%) relative humidity (RH). From the IR data, it is shown that d-(AAAAATTTTT)2 and poly(dA).poly(dT) adopt a B-genus conformation in films with high water content. When the relative humidity of the film is decreased, the IR spectra reflect a gradual evolution of the geometry of both d(AAAAATTTTT)2 and poly(dA).poly(dT) into a form intermediate between the B genus and A genus, but the IR spectrum of a pure A genus has not been obtained. In these DNAs at 75% RH, the IR bands of adenosine have the same frequencies as those found in poly(dA-dT) at 75% RH where the local furanose conformation is C3' endo/anti, but the thymidine frequencies do not resemble those of poly(dA-dT) at 75% RH but rather those of poly(dA-dT) at high humidities. It is concluded that both poly(dA).poly(dT) and d(AAAAATTTTT)2 adopt a fully heteronomous duplex geometry in cast films at low humidity. For studies in aqueous solution the Raman effect was employed. As a model for the heteronomous conformation in solution, the duplex poly(rA).poly(dT) was used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the hydration of the minor groove of A.T synthetic DNA polymers by volume and heat changes

The minor-groove ligand netropsin provides a sensitive probe of the hydration difference between poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)]. We have measured the volume change delta V accompanying binding of netropsin to these polymers, using an improved magnetic suspension densimeter. For poly(dA).poly(dT) we find delta V = +97 mL/mol of bound netropsin at pH 7.0 and 10 mM sodium phosphate buffer. For poly[d(AT)].poly[d(AT)] we find delta V = -16 mL/mol of bound netropsin. This striking differential effect suggests that the poly(dA).poly(dT) duplex compresses more water (or is more extensively hydrated). From our enthalpy and entropy results we estimate the approximately 10 water molecules, immobilized in the minor groove of this system, are displaced by each netropsin bound. The volume increase, however, is substantially larger than can be explained by a simple melting of these immobilized water molecules in the minor groove. A decompression of at least 40 water molecules must attend the complexation to the poly(dA).poly(dT) duplex. This suggests that the conformation change attending the binding of the drug to this polymer duplex causes a further dehydration, whereas no such change in dehydration and configuration for the heteropolymer system is indicated.     

The poly dA strand of poly dA.poly dT adopts an A-form in solution: a UV resonance Raman study.

The study by resonance Raman spectroscopy with a 257 nm excitation wave-length of adenine in two single-stranded polynucleotides, poly rA and poly dA, and in three double-stranded polynucleotides, poly dA.poly dT, poly(dA-dT).poly(dA-dT) and poly rA.poly rU, allows one to characterize the A-genus conformation of polynucleotides containing adenine and thymine bases. The characteristic spectrum of the A-form of the adenine strand is observed, except small differences, for poly rA, poly rA.poly rU and poly dA.poly dT. Our results prove that it is the adenine strand which adopts the A-family conformation in poly dA.poly dT.     

Oligopurine.oligopyrimidine tracts do not have the same conformation as analogous polypurine.polypyrimidines

The ability of tracts of synthetic oligopurine.oligopyrimidines containing both adenosine and guanosine residues to approach the conformation of analogous polypurine.polypyrimidines has been examined as a function of tract length by CD spectroscopy. Tracts of up to 19 contiguous, alternating dA and dG residues yield CD spectra that are distinctly different from that of the analogous alternating polymer. Thus the structural changes reflected in the unusual CD spectrum of poly[d(AG)].poly[d(CT)] must require even longer tract lengths. Tracts of contiguous adenosines flanked by guanosine residues were seen to approach the CD spectrum of poly[dA].poly[dT] quite slowly as a function of tract length, requiring more than 24 contiguous adenosines to give CD spectra similar to the homopolymer. These results lead us to the conclusion that oligopurine tracts in vivo are not well modeled by synthetic polypurine.polypyrimidines with one or two base pair repeating units.     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA).poly(dT) and poly(dG).poly(dC), and with triple helical poly(dA).[poly(dT)](2) and poly(dC).poly(dG).poly(dC)(+) were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA).poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG).poly(dC) and -poly(dC).poly(dG).poly(dC)(+) complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

Vibrational circular dichroism studies of the A-to-B conformational transition in DNA.

The vibrational circular dichroism (VCD) spectra of several natural DNAs as well as tRNA, poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) are reported for the base deformation modes in the IR region from 1700 to 1550 cm-1 for the polymers in D2O as well as in high alcohol dehydrating conditions. Spectra of both the B- and A-forms were identified. The A-form DNA VCD, not previously reported, has characteristics that can be found in the VCD spectra of RNAs as would be expected from the similarity of their structures. The VCD is sequence-dependent. Under the dehydrating conditions studied, poly(dA-dT)poly(dA-dT),poly(dA).poly(dT), and a high-A-T fraction natural DNA had a different bandshape from the other DNAs, which was similar to that of poly(rA).poly(rU). Poly(dG-dC).poly-(dG-dC) did not form an A-form in high-alcohol conditions but instead had a VCD spectrum much like that of its high-salt-induced Z-form. Qualitative differences seen experimentally between A- and B-form DNA VCD were suggested by the differences in the coupled oscillator VCD calculated for the two forms.     

Investigation of DNA Binding Modes for a Symmetrical Cyanine Dye Trication: Effect of DNA Sequence and Structure

Abstract

The DNA binding behavior of a tricationic cyanine dye (DiSC₃₊(5)) was studied using the [Poly(dA-dT)]₂, [Poly(dI-dC)]₂ and Poly(dA)?Poly(dT) duplex sequences and the Poly(dA) ?2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA)?Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]₂ duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]₂ sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J.L., et al. (1999) J. Am. Chem. Soc. 121, 2987–2995] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.     

Binding geometries of triple helix selective benzopyrido [4,3-b]indole ligands complexed with double- and triple-helical polynucleotides

The binding modes of three benzopyrido [4,3-b]indole derivatives (and one benzo[-f]pyrido [4-3b] quinoxaline derivative) with respect to double helical poly(dA) · poly(dT) and poly[d(A-T)]₂ and triple-helical poly(dA) · 2poly(dT) have been investigated using linear dichroism (LD) and CD: (I) 3-methoxy-11-amino-BePI where BePI = (7H-8-methyl-benzo[e]pyrido [4,3-b]indole), (II) 3-methoxy-11-[(3′-amino) propylamino]-BePI, (III) 3-methoxy-7-[(3′-diethylamino)propylamino] BgPI where BgPI = (benzo[g]pyrido[4,3-b]indole), and (IV) 3-methoxy-11-[(3′-amino)propylamino] B f P Q where B f P Q = {benzo[-f]pyrido[4-3b]quinoxaline}. The magnitudes of the reduced LD of the electronic transitions of the polynucleotide bases and of the bound ligands are generally very similar, suggesting an orientation of the plane of the ligands' fused-ring systems preferentially perpendicular to the helix axis. The LD results suggest that all of the ligands are intercalated for all three polynucleotides. The induced CD spectrum of the BePI chromophore in the (II-BePI)-poly[d(A-T)]₂ complex is almost a mirror image of that for the (I-BePI)-poly(dA) · poly(dT) and (I-BePI)-poly(dA) · 2poly(dT) complexes, suggesting an antisymmetric orientation of the BePI moiety upon intercalation in poly[d(A-T)]₂ compared to the other polynucleotides. The induced CD of I-BePI bound to poly(dA) · 2poly(dT) suggests a geometry that is intermediate between that of its other two complexes. The concluded intercalative binding as well as the conformational variations between the different BePI complexes are of interest in relation to the fact that BePI derivatives are triplex stabilizers. © 1997 John Wiley & Sons, Inc. Biopoly 42: 101–111, 1997     

Sequence and length dependent thermodynamic differences in heterocyclic diamidine interactions at AT base pairs in the DNA minor groove

With the goal of developing a better understanding of the antiparasitic biological action of DB75, we have evaluated its interaction with duplex alternating and nonalternating sequence AT polymers and oligomers. These DNAs provide an important pair of sequences in a detailed thermodynamic analysis of variations in interaction of DB75 with AT sites. The results for DB75 binding to the alternating and nonalternating AT sequences are quite different at the fundamental thermodynamic level. Although the Gibbs energies are similar, the enthalpies for DB75 binding with poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) are +3.1 and -4.5 kcal/mol, respectively, while the binding entropies are 41.7 and 15.2 cal/mol.K, respectively. The underlying thermodynamics of binding to AT sites in the minor groove plays a key role in the recognition process. It was also observed that DB75 binding with poly(dA).poly(dT) can induce T.A.T triplet formation and the compound binds strongly to the dT.dA.dT triplex.     

Interaction Specificity of Benzimidazol Group Compounds with AT-Containing Polynucleotides

Abstract

The interaction between polynucleotides: poly(dA)-poly(dT), poly(dA-dT), poly(am2dA- dT), and the AT-specific compounds of benzimidazol group has been studied. It is been shown that these compounds bind to poly(dA)-poly(dT) and poly(dA-dT) at low and high salt concentration in solution. Poly(am2dA-dT) interacts with AT-specific compounds only at low salt, where this polynucleotide is in a B-form, but not at high salt when the polynucleotide converts to another conformation. Thus, the interaction specificity of the groove-binding ligands is influenced not only by the minor groove substituents, but the peculiarities of the secondary structure of polynucleotides.     

Effect of cesium on the volume of the helix-coil transition of dA.dT polymers and their ligand complexes

The pressure dependence of the helix–coil transition of poly(dA)∙poly(dT) and poly[d(A-T)]·poly[d(A-T)] in aqueous solutions of NaCl and CsCl at concentrations between 10 and 200 mM is reported and used to calculate the accompanying volume change. We also investigated the binding parameters and volume change of ethidium bromide binding with poly(dA)∙poly(dT) and poly[d(A-T)]·poly[d(A-T)] in aqueous solutions of these two salts. The volume change of helix–coil transition of poly(dA)∙poly(dT) in Cs⁺-containing solutions differs by less than 1 cm³ mol^− 1 from the value measured when Na⁺ is the counter-ion. We propose that this insensitivity towards salt type arises if the counter-ions are essentially fully hydrated around DNA and the DNA conformation is not significantly altered by salt types. Circular dichroism spectroscopy showed that the previously observed large volumetric disparity for the helix–coil transition of poly[d(A-T)]·poly[d(A-T)] in solutions containing Na⁺ and Cs⁺ is likely result of a Cs⁺-induced conformation change that is specific for poly[d(A-T)]·poly[d(A-T)]. This cation-specific conformation difference is mostly absent for poly(dA)∙poly(dT) and EB bound poly[d(A-T)]·poly[d(A-T)].     

Two binding modes of netropsin are involved in the complex formation with poly(dA-dT).poly(dA-dT) and other alternating DNA duplex polymers

Using CD measurements we show that the interaction of netropsin to poly(dA-dT).poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA.dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA).poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT).poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.