Sequence-dependent variation in the conformation of DNA

The specificity of action of the enzyme DNAase I on double-stranded DNA polymers of defined sequence has been investigated. The results obtained with the alternating copolymers poly[d(A-T)] · poly[d(A-T)] and poly[d(G-C)] · poly[d(G-C)] support the suggestion of Klug et al. (1979) that regions of double-stranded DNA containing alternating purine-pyrimidine sequences may exist as structural variants of the classical B-form under physiological salt conditions. Digestion of defined oligomers containing alternating dG-dC sequences indicate that these too exist in some “alternating-B” structure in solution under similar conditions. The results obtained with the oligomers also provide a number of insights into the mode of action of DNAase I.In the case of the B-DNA dodecamer d(C-G-C-G-A-A-T-T-C-G-C-G), for which the crystal structure has been solved (Dickerson &; Drew, 1981), there is a very good correlation between the sites of rapid DNAase I cutting and positions of high local helical twist.     

Differences in melting behavior between homopolymers and copolymers of DNA: Role of nonbonded forces for GC and the role of the hydration spine and premelting transition for AT

Melting measurements of the mono-base-pair DNA polymers showed that the melting temperature T_m of the B-DNA homopolymer poly (dA ) · poly (dT) is higher than that of the copolymer poly [d(A-T)]. On the other hand, the T_mof the B-DNA homopolymer poly (dG) · poly (dC) is lower than that of the copolymer poly [d (G-C)]. From a structural point of view, the cross-strand base-stacking interaction in a DNA homopolymer is weaker than that in a DNA copolymer with the same base pair. One would then expect that all the DNA homopolymers are less stable than the copolymer with the same base pair. We find that the inversion of the melting order seen in the AT mono-base-pair DNA polymers is caused by the enhanced thermal stability of poly (dA) · poly (dT) from a well-defined spine of hydration attached to its minor groove. In this paper we employ the modified self-consistent phonon theory to calculate base-pair opening probabilities of four B-DNA polymers: poly(dA)-poly(dT), poly(dG) · poly(dC), poly[d(A-T)], and poly[d(G-C)] at temperatures from room temperature through the melting regions. Our calculations show that the spine of hydration can give the inverted melting order of the AT polymers as compared to the GC polymers in fair agreement with experimental measurements. Our calculated hydration spine disruption behavior in poly(dA) · poly(dT) at premelting temperatures is also in agreement with experimentally observed premelting transitions in poly (dA) · poly (dT). The work is in a sense a test of the validity of our models of nonbonded interactions and spine of hydration interactions. We find we have to develop the concept of a strained bond to fit observations in poly (dA) · poly(dT). The strained-bond concept also explains the otherwise anomalous stability of the hydration chain. © 1993 John Wiley & Sons, Inc.     

Binding of ligands to a one-dimensional heterogeneous lattice. III. Kinetic model and relaxation study of the interaction of tilorone with DNA and polynucleotides

A temperature-jump relaxation study of the interaction of tilorone with different polynucleotides and DNA has been performed. A single relaxation time, attributed to the intercalation step, has been observed in the case of poly[d(A-T)]·poly[d(A-T)], poly[d(A-C)]·poly[d(G-T)], poly[d(G-C)]·poly[d(G-C)], and poly(dG)·poly(dC). No intercalation into poly(dA)·poly(dT) occurs, and the interaction with poly(dG)·poly(dC) is different from what is observed with the other intercalating homopolymers. Refinement of the binding model is suggested from the analysis of the kinetic data. The relaxation curves obtained with DNA are well simulated based on a binding mechanism where DNA is considered a heterogeneous lattice and each type of site behaves as if it were located in the corresponding homopolymer. Poly(dA)·poly(dT) shows a unique behavior: studies of the effects of concentration and temperature indicate that tilorone acts as a probe of a process involving the polynucleotide alone. This process appears to be related to the dynamic structure of the nucleic acid and is detectable only when the bound dye is not intercalated.     

The analysis of circular dichroism spectra of natural DNAs using spectral components from synthetic DNAs

We have tested 21 different basis sets of synthetic DNA circular dichroism spectra and have slected one for use in spectral analyses of natural DNAs. This “standard” set consists of spectra of eight polymers: poly[d(A-A-T)·d(A-T-T)], poly[d(A-G-G)·d(C-C-T)], poly[d(A-T)·d(A-T)], poly[d(G-C)·d(G-C)], poly[d(A-G)·d(C-T)], poly[d(A-C)·d(G-T)], poly[d(A-T-C)·d(G-A-T)], and poly[d(A-G-C)·d(G-C-T)]. This basis set, applied according to the first-neighbor polymer procedure of Gray and Tinoco, allows a more uniformly accurate spectral analysis of six natural complex DNAs and eight (A+T)-rich satellite DNAs for base composition and first-neighbor frequencies than was previously possible. We find that spectra of poly[d(A)·d(T)] and/or poly[d(A-C-T-)·d(A-G-T)] are not generally required for good analysis results but we show in this and the following paper that these spectra are needed for the most accurate analyses of some satellite DNAs.     

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.     

A molecular mechanical study of netropsin-DNA interactions

We present molecular mechanical calculations on the complexes of netropsin with dA₆·dT₆, d(TATATA)₂, d(CGCGCG)₂, and d(CGCGAATTCGCG)₂. The complexes were model built using computer graphics and then completely energy refined. Our calculations are consistent with the observed AT preference for netropsin and suggest that mixed sugar pucker geometries should be more stable than uniform in netropsin complexes with poly[d(A-T)]·poly[d(A-T)] and poly(dA)·poly(dt). The netropsin·d(TATATA) and netropsin·dA₆·dT₆ complexes are significantly different in structure, leading to a possible reason why the observed thermodynamics of netropsin-association with poly[d(A-T)]·poly[d(A-T)] and with poly(dA)·poly(dT) are so different. We also model built and energy refined a structure of netropsin-d(CGCGAATTCGCG)₂ using as a guide the nmr data of Patel [(1982) Proc. Natl. Acad. Sci. USA, 79 , 6424–6428] and found a three-dimensional structure qualitatively consistent with the NOE enhancements observed by him. After our calculations were completed, we learned of an x-ray structure of a netropsin:d(CGCGAATTCGCG)₂ complex, and we compared the structure found in our calculation with the x-ray structure.     

DNA binding of a spermine derivative: Spectroscopic study of anthracene-9-carbonyl-n1-spermine with poly[d(G-C)·(d(G-C))] and poly[d(A-T) · d(A-T)]

The binding of polyamines, including spermidine ( 1 ) and spermine ( 2 ), to poly[d(G-C) · d(G-C) ] was probed using spectroscopic studies of anthracene-9-carbonyl-N¹-spermine ( 3 ); data from normal absorption, linear dichroism (LD), and circular dichroism (CD) are reported. Ligand LD and CD for transitions located in the DNA region of the spectrum were used. The data show that 3 binds to DNA in a manner characteristic of both its amine and polycyclic aromatic parts. With poly [(dG-dC) · (dG-dC)], binding modes are occupied sequentially and different modes correspond to different structural perturbations of the DNA. The most stable binding mode for 3 with poly[d(G-C) · d(G-C)] has a site size of 6 ± 1 bases, and an equilibrium binding constant of (2.2 ± 1.1) × 10⁷ M^?1 with the anthracene moiety intercalated. It dominates the spectra from mixing ratios of approximately 133:1 until 6:1 DNA phosphate: 3 is reached. The analogous data for poly [d(A-T) · d(A-T)] between mixing ratios 36:1 and 7:1 indicates a site size of 8.3 ± 1.1 bases and an equilibrium binding constant of (6.6 ± 3.3) × 10⁵ M^?1. Thus, 3 binds preferentially to poly [d(G-C) · d(G-C)] at these concentrations. © 1994 John Wiley & Sons, Inc.     

Sequence-dependant polymorphism of DNA duplex in solutions

Raman spectra of six synthetic polydeoxyribonucleotide duplexes with different base sequences have been examined in aqueous solutions with different salt or nucleotide concentrations. Detailed conformational differences have been indicated between B and Z forms of poly[d(G-C)] X poly[d(G-C)], between B forms of poly[d(G-C)] X poly[d(G-C)] and poly[d(G-m5C)] X poly[d(G-m5C)], between A and B forms of poly(dG) X poly(dC), between B and "CsF" forms of poly[d(A-T)] X poly[d(A-T)], between B forms of poly[d(A-U)] X poly[d(A-U)] and poly[d(A-T)] X poly[d(A-T)], and between low- and high-salt (CsF) forms of poly(dA) X poly(dT). The Raman spectrum of calf-thymus DNA in aqueous solution was also observed and was compared with the Raman spectra of its fibers in A, B, and C forms.     

Template specificities of Aclacinomycin B on the inhibition of DNA-dependent RNA synthesis in vitro

Summary The effect of Aclacinomycin B (ACM-B), an anthracycline antitumor antibiotic, on the DNA-dependent RNA synthesis using single- and double-stranded DNAs of known base content and sequence is studied. The data show that ACM-B effectively inhibits the double-stranded DNA-directed RNA synthesis with a preference of poly[d(A-T)] > poly[d(G-C)] > poly[d(I-C)]. In contrast, it has no inhibitory effect on the template function of single-stranded DNA (e.g. poly dA, poly dT, and poly dC). These results suggest that the mechanism of ACM-13 inhibition, like other anthracycline antibiotics, is by intercalation. In addition to the base specificity, there are also dramatic differences in inhibition depending on the base sequence in the DNA template. Thus, ACM-13 preferentially inhibits the alternating double-stranded copolymers over the double-stranded homopolymers; e.g. poly [d(A-T)] is inhibited to a greater extent than poly dA · poly dT and poly [d(G-C)] is inhibited more than poly dG · poly dC. Since the inhibition by ACM-13 can be totally abolished when assayed in excess amount of DNA, this result suggests that ACM-B inhibition of RNA synthesis is solely on the DNA template (which is in support of the intercalation model), and has ruled out the possibility that ACM-B may also exert an inhibitory effect on the activity of RNA polymerase per se.     

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     

Conformational properties of poly[d(G-T)].poly[d(C-A)] and poly[d(A-T)] in low- and high-salt solutions: NMR and laser Raman analysis

³¹P- and ¹H-nmr and laser Raman spectra have been obtained for poly[d(G-T)]·[d(C-A)] and poly[d(A-T)] as a function of both temperature and salt. The ³¹P spectrum of poly[d(G-T)]·[d(C-A)] appears as a quadruplet whose resonances undergo separation upon addition of CsCl to 5.5M. ¹H-nmr measurements are assigned and reported as a function of temperature and CsCl concentration. One dimensional nuclear Overhauser effect (NOE) difference spectra are also reported for poly[d(G-T)]·[d(C-A)] at low salt. NOE enhancements between the H8 protons of the purines and the C5 protons of the pyrimidines, (H and CH₃) and between the base and H-2′,2″ protons indicate a right-handed B-DNA conformation for this polymer. The NOE patterns for the TH3 and GH1 protons in H₂O indicate a Watson–Crick hydrogen-bonding scheme. At high CsCl concentrations there are upfield shifts for selected sugar protons and the AH2 proton. In addition, laser Raman spectra for poly[d(A-T)] and poly[d(G-T)]·[d(C-A)] indicate B-type conformations in low and high CsCl, with predominantly C2′-endo sugar conformations for both polymers. Also, changes in base-ring vibrations indicate that Cs⁺ binds to O2 of thymine and possibly N3 of adenine in poly[d(G-T)]·[d(C-A)] but not in poly[d(A-T)]. Further, ¹H measurements are reported for poly[d(A-T)] as a function of temperature in high CsCl concentrations. On going to high CsCl there are selective upfield shifts, with the most dramatic being observed for TH1′. At high temperature some of the protons undergo severe changes in linewidths. Those protons that undergo the largest upfield shifts also undergo the most dramatic changes in linewidths. In particular TH1′, TCH₃, AH1′, AH2, and TH6 all undergo large changes in linewidths, whereas AH8 and all the H-2′,2″ protons remain essentially constant. The maximum linewidth occurs at the same temperature for all protons (65°C). This transition does not occur for d(G-T)·d(C-A) at 65°C or at any other temperature studied. These changes are cooperative in nature and can be rationalized as a temperature-induced equilibrium between bound and unbound Cs⁺, with duplex and single-stranded DNA. NOE measurements for poly[d(A-T)] indicate that at high Cs⁺ the polymer is in a right-handed B-conformation. Assignments and NOE effects for the low-salt ¹H spectra of poly[d(A-T)] agree with those of Assa-Munt and Kearns [(1984) Biochemistry 23 , 791–796] and provide a basis for analysis of the high Cs⁺ spectra. These results indicate that both polymers adopt a B-type conformation in both low and high salt. However, a significant variation is the ability of the phosphate backbone to adopt a repeat dependent upon the base sequence. This feature is common to poly[d(G-T)]·[d(C-A)], poly[d(A-T)], and some other pyr–pur polymers [J. S. Cohen, J. B. Wouten & C. L Chatterjee (1981) Biochemistry 20 , 3049–3055] but not poly[d(G-C)].     

A hydrogen exchange study of the open segment in a DNA double helix

The kinetics of the hydrogen-deuterium exchange reactions of double-helical poly[d(A-T)]·poly[d(A-T)], poly(dA)·poly(dT), and constituent nucleosides (deoxyadenosine and thymidine) have been examined at various temperatures by stopped-flow ultraviolet spectrophotometry, in the spectral region 240–300 nm. The results were interpreted on the basis of a mechanism of the hydrogen exchange reaction of a helical polynucleotide, proposed by Englander and colleagues as well as by the Tsuboi and Nakanishi group. It was concluded that the rates of the base-pair opening reactions are nearly equal to one another in double-helical DNAs, irrespective of the base sequence. On the other hand, the free energy required for bringing the open segment at a particular base-pair was found to be much greater for poly(dA)·poly(dT) than for poly[d(A-T)]· poly[d(A-T)].     

CD studies of double-stranded polydeoxynucleotides composed of repeating units of contiguous homopurine residues

Double-stranded synthetic polydeoxynucleotides of the general form poly[d(G_nC_n)] · poly[d(G_nC_n)], poly[d(G_nC)] · poly[d(GC_n)], and poly[d(A_nT_n)] · poly[d(A_nT_n)] have been synthesized. When n = 4 or larger, the CD spectra of polymers of the form poly[d(G_nC_n)] · poly[d(G_nC_n)] or poly[d(G_nC)] · poly[d(GC_n)] closely resemble the spectrum of poly[dG] · poly[dC], suggesting that a string of four continguous guanosine residues is sufficient to induce a conformation resembling that of the polypurine · polypyrimidine. With polymers of the form poly[d(A_nT_n)] · poly[d(A_nT_n)], however, the CD spectrum only gradually approaches that of poly[dA] · poly[dT].     

Spectroscopic studies of 9-hydroxyellipticine binding to DNA

The binding of 9-hydroxyellipticine to calf thymus DNA, poly[d(A-T)]₂, and poly-[d(G-C)]₂ has been studied in detail by means of CD, linear dichroism, resonance light scattering, and molecular dynamics. The transition moment polarizations of 9-hydroxyelliptiycine were determined in polyvinyl alcohol stretched film. Spectroscopic solution studies of the DNA/drug complex are combined with theoretical CD calculations using the final 50 ps of a series of molecular dynamics simulations as input. The spectroscopic data shows 9-hydroxyellipticine to adopt two main binding modes, one intercalative and the other a stacked binding mode involving the formation of drug oligomers in the DNA major groove. Analysis of the intercalated binding mode in poly[d(A-T)]₂ suggests the 9-hydroxyellipticine hydroxyl group lies in the minor groove and hydrogen bonds to water with the pyridine ring protruding into the major groove. The stacked binding mode was examined using resonance light scattering and it was concluded that the drug was forming small oligomer stacks rather than extended aggregates. Reduced linear dichroism measurements suggested a binding geometry that precluded a minor groove binding mode where the plane of the drug makes a 45° angle with the plane of the bases. Thus it was concluded that the drug stacks in the major groove. No obvious differences in the mode of binding of 9-hydroxyellipticine were observed between different DNA sequences; however, the stacked binding mode appeared to be more favorable for calf thymus DNA and poly[d(G-C)]₂ than for poly[d(A-T)]₂, an observation that could be explained by the slightly greater steric hindrance of the poly[d(A-T)]₂ major groove. A strong concentration dependence was observed for the two binding modes where intercalation is favored at very low drug load, with stacking interactions becoming more prominent as the drug concentration is increased. Even at DNA : drug mixing ratios of 70:1 the stacked binding mode was still important for GC-rich DNAs. © 1998 John Wiley & Sons, Inc. Biopoly 46: 127–143, 1998     

Pressure dependence of the helix–coil transition temperature of poly[d(G-C)]

The Pressure Dependence of the Helix-Coil Transition Temperature (T_m) of Poly[d(G-C)] was studied as a function of sodium ion concentration in phosphate buffer. The molar volume change of the transition (ΔV) was calculated using the Clapeyron equation and calorimetrically determined enthalpies. The ΔV of the transition increased from +4.80 (±0.56) to +6.03 (±0.76) mL mol^?1 as the sodium ion concentration changed from 0.052 to 1.0M. The van't Hoff enthalpy of the transition calculated from the half-width of the differentiated transition displayed negligible pressure dependence: however, the value of this parameter decreased with increasing sodium ion concentration, indicating a decrease in the size of the cooperative unit. The volume change of the transition exhibits the largest magnitude of any double-stranded DNA polymer measured using this technique. For poly[d(G-C)] the magnitude of the change in ΔV with sodium ion concentration (0.94 ± 0.05 mL mol^?1) is approximately one-half that observed for either poly[d(A-T)] or poly (dA)·poly(dT). The ΔV values are interpreted as arising from changes in the hydration of the polymer due to the release of counterions and changes in the stacking of the bases of the coil form. As a consequence of solvent electrostriction, the release of counterions makes a net negative contribution to the total ΔV, implying that disruption of the slacking interactions contributes a positive volume change to the total ΔV. The larger magnitude of the ΔV compared with that of other double-stranded polymers may be due in part to the high helix-coil transition temperature of poly[d(G-C)], which will attenuate the contribution of electrostriction to the total volume change. The data in addition show that in the absence of other cellular components, the covalent structure of DNA is stabile under conditions of temperature and pressure more extreme than those experienced by any known organism. © 1995 John Wiley & Sons, Inc.     

Natural DNA sequences can form left-handed helices in low salt solution under conditions of topological constraint

The conformation of DNA that originates from association of complementary single-stranded circles (form V DNA) is investigated in solution at low salt concentration. It is shown that circular dichroism extended to the far ultraviolet region (down to 165 nm) represents a powerful tool for determination of the handedness of double helical DNAs in solution. The positive intense band at 186 nm followed by a strong negative band around 170 nm is characteristic of all right-handed helical forms (B,A) of DNA, whereas the circular dichroism spectrum of the Z form of poly[d(G-C)] of opposite helical sense represents a quasi inversion of these far ultraviolet bands. Thus, form V DNA is found to represent a co-existence of left-handed Z-type and right-handed B double helical stretches in addition to negative superturns. The Raman spectrum of form V DNA provides further support for the contribution of a left-handed double helical conformation, as shown by comparison to the high resolution Raman spectra of poly[d(G-C)] in the Z and B forms.The analysis of present spectroscopic data and the analysis of occurrence of alternating [d(G-C)] purine-pyrimidine sequences in the form V DNA used strongly suggest that in DNA of natural sequence, topological constraint may generate left-handed double helices, a conformation thought so far to be limited to the alternating [d(G-C)] sequences. Such structure could play a role in recognition and regulation of gene expression.     

Study of conformation characteristics of polydeoxyribonucleotides with non-random base sequence by slow 1H----3H exchange

The rate constants of 1H----3H exchange between water and C8H-groups of purinic residues of alternating polynucleotides: poly[d(A-T)].poly[d(A-T)] (I), poly[d(G-C)].poly[d(G-C)] (II), poly[d(A-C)].poly[d(G-T)] (III) and homopolynucleotides: poly(dA).poly(dt) (IV), poly(dG).poly(dC) (V), as well as DNA E. coli, was determined in 0.15 M NaCl at 25 degrees C. The retardation of exchange observed at these conditions (compared to that of the B-form DNA) is in agreement with the model of B-alternating structure for the (I) and is attributed to the co-existence of B- and A-conformers for the (V) in solution. Absence of distinguishable differences in exchange rate constants for purinic residues of the (II), (III) and (IV) (compared to that of the B-form DNA) evidences that conformations of these polynucleotides in solution are similar to "canonical" B-form DNA and don't correlate with the model of "heteronomous" DNA which was proposed for (IV).     

Infrared dichroism of polynucleotides of repeated sequence. I. Poly(dA)·poly(dT) and poly[d(A-T)]·poly[d(A-T)]

Infrared dichroism measurements of oriented films of poly(dA)·poly(dT) and poly[d(A-T)]·poly[d(A-T)] have been made under the conditions of low salts content and high humidity for which the geometry is known. The angles which the transition moments make with the helix axis are compared with the orientations of the corresponding bonds. Except for the in-plane base model of poly[(A-T)]·poly[d(A-T)], there is no agreement. This may imply either that a model which assumes bonds and transition moments to be colinear is not acceptable or that x-ray data are inaccurate. These possibilities are discussed especially with respect to phosphate group orientation. An appendix gives the derivations of dichroic-ratio expressions for helical molecules of different symmetry types.     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

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)].