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.     

Mercury-induced transitions between right-handed and putative left-handed forms of poly[d(A-T).d(A-T)] and poly[d(G-C).d(G-C)]

Poly[d(A-T).d(A-T)] and poly[d(G-C).d(G-C)], each dissolved in 0.1 M NaClO4, 5 mM cacodylic acid buffer, pH 6.8, experience inversion of their circular dichroism (CD) spectrum subsequent to the addition of Hg(ClO4)2. Let r identical to [Hg(ClO4)2]added/[DNA-P]. The spectrum of the right-handed form of poly[d(A-T).d(A-T)] turns into that of a seemingly left-handed structure at r greater than or equal to 0.05 while a similar transition is noted with poly[d(G-C).(G-C)] at r greater than or equal to 0.12. The spectral changes are highly cooperative in the long-wavelength region above 250 nm. At r = 1.0, the spectra of the two polymers are more or less mirror images of their CD at r = 0. While most CD bands experience red-shifts upon the addition of Hg(ClO4)2, there are some that are blue-shifted. The CD changes are totally reversible when Hg(II) is removed from the nucleic acids by the addition of a strong complexing agent such as NaCN. This demonstrates that mercury keeps all base pairs in register.     

Binding mode of porphyrins to poly[d(A-T)(2)] and poly[d(G-C)(2)

We examined the binding geometry of Co-meso-tetrakis (N-methyl pyridinium-4-yl)porphyrin, Co-meso-tetrakis (N-n-butyl pyridinium-4-yl)porphyrin and their metal-free ligands to poly[d(A-T)(2)] and poly[d(G-C)(2)] by optical spectroscopic methods including absorption, circular and linear dichroism spectroscopy, and fluorescence energy transfer technique. Signs of an induced CD spectrum in the Soret band depend only on the nature of the DNA sequence; all porphyrins exhibit negative CD when bound to poly[d(G-C)(2)] and positive when bound to poly[d(A-T)(2)]. Close analysis of the linear dichroism result reveals that all porphyrins exhibit outside binding when complexed with poly[d(A-T)(2)], regardless of the existence of a central metal and side chain. However, in the case of poly[d(G-C)(2)], we observed intercalative binding mode for two nonmetalloporphyrins and an outside binding mode for metalloporphyrins. The nature of the outside binding modes of the porphyrins, when complexed with poly[d(A-T)(2)] and poly[d(G-C)(2)], are quite different. We also demonstrate that an energy transfer from the excited nucleo-bases to porphyrins can occur for metalloporphyrins.     

Circular dichroism spectra of poly[d(AC):d(GT)], poly[r(AC):r(GU)], and hybrids poly[d(AC):r(GU)] and poly[r(AC):d(GT)] in the presence of ethanol.

Ultraviolet circular dichroism spectra have been obtained in aqueous solutions in the presence and absence of ethanol for a synthetic DNA, poly[d(AC):d(GT)], a synthetic RNA, poly[r(AC):r(GU)], and two DNA:RNA hybrids, poly[d(AC):r(GU)] and poly[r(AC):d(GT)]. In the absence of ethanol, we find that the RNA and DNA spectra are dissimilar, while the spectra of the hybrids show differing degrees of similarity to that of the RNA. In the presence of 60–80% ethanol by weight, the spectra of the DNA and both hybrids become much closer to the spectrum of the RNA, which remains relatively unchanged. We interpret the results as indicating that DNA can undergo a change to an A-type conformation in the presence of ethanol and that the DNA:RNA hybrids are not wholly restricted to an RNA-like conformation in the absence of ethanol.     

Right- and left-handed helixes of poly[d(A-T)].poly[d(A-T)] investigated by infrared spectroscopy

The secondary structures of double-stranded poly[d(A-T)].poly[d(A-T)] in films have been studied by IR spectroscopy with three different counterions (Na+, Cs+, and Ni2+) and a wide variety of water content conditions (relative humidity between 100 and 47%). In addition to the A-, B-, C-, and D-form spectra, a new IR spectrum has been obtained in the presence of nickel ions. The IR spectra of Ni2+-poly[d(A-T)].poly[d(A-T)] films are analyzed by comparison with previously assigned IR spectra of left-handed poly[d(G-C)].poly[d(G-C)] and poly[d(A-C)].poly[d(G-T)], and it is possible to conclude that they reflect a Z-type structure for poly[d(A-T)].poly[d(A-T)]. The Z conformation has been favored by the high polynucleotide concentration, by the low water content of the films, and by specific interactions of the transition metal ions with the purine bases stabilized in a syn conformation. A structuration of the water hydration molecules around the double-stranded Ni2+-poly[d(A-T)].poly[d(A-T)] is shown by the presence of a strong sharp water band at 1615 cm-1.     

Assignment of resonances in the phosphorus-31 nuclear magnetic resonance spectrum of poly[d(A-T)] from phosphorothioate substitution

Two phosphorothioate analogues of poly[d(A$-T)] have been synthesized enzymatically. In one, poly[d(A$-T)], dTMP is replaced by thymidine 5'-O-phosphorothioate; in the other, poly[d(T$-A)], dAMP is replaced by 2'-deoxyadenosine 5'-O-phosphorothioate. The 31P NMR spectrum of poly[d-(A-T)] in solutions at low salt concentration shows two resonances at 51.80 and -4.25 ppm relative to trimethyl phosphate. The corresponding values for poly[d(T$-A)] are 51.51 and -4.43 ppm. These data allow the assignment of the downfield resonance at -4.23 ppm in poly[d(A-T)] to the phosphate group of d(TpA) and the resonance at -4.41 ppm to that of d(ApT). Thus, strong evidence is provided for a repeating dinucleotide structure. A comparison of the 31P NMR spectra of the various polymers in solutions of 2 M CsF reveals that both resonances are shifted upfield by approximately 0.9 ppm in the case of the phosphorothioates and by 0.2 or 0.4 ppm in the case of the phosphates. An upfield shift of about 0.18 ppm can also be observed for the two corresponding dinucleoside monophosphates. Thus, the upfield shift induced by high concentrations of CsF is not specific for the polymer backbone.     

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)].     

Effect of 5-alkyl substitution of uracil on the thermal stability of poly [d(A-r5U)] copolymers.

The thermal transition of poly[d(A-r5U)] polydeoxynucleotides (where r was a hydrogen atom, or a methyl, ethyl, n-propyl, n-butyl or n-pentyl group) was studied by measuring the derivative melting profiles of the polymers in the range of 0.01--0.36 M K+, at pH 6.8. According to the Tm values, polydeoxynucleotide analogues show lower thermal stability than poly[d(A-T)] at any counterion concentration applied. At a given salt concentration, Tm of the alkyl analogues decreased as the number of carbon atoms (n) in the r substituent of poly[d(A-r5U)] increased. 1/Tm plotted against against 1/n yielded a linear relationship. Cooperativity of the melting of all poly[d(A--U)] analogues decreased with the increase of salt concentration in the solution. This change depended again on 5-substitution of the uracil moiety of poly[d(A-U)]. Smallest decrease was observed in the case of poly[d(A--U)] whereas largest decrease was shown by poly[d(A-pe5U)] (pe=pentyl group).     

A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands.

The vacuum UV CD spectra of GpC, CpG, GpG, poly[r(A)], poly[r(C)], poly[r(U)], poly[r(A-U)], poly[r(G).r(C)], poly[r(A).r(U)], and poly[r(A-U).r(A-U)] were measured down to at least 174 nm. These spectra, together with the published spectra of poly[r(G-C).r(G-C)], CMP, and GMP, were sufficient to estimate the CD changes upon base pairing for four double-stranded RNAs. The vacuum UV CD bands of poly[r(A)], poly[r(C)], and the dinucleotides GpC and CpG were temperature dependent, suggesting that they were due to intrastrand base stacking. The dinucleotide sequence isomers GpC and CpG had very different vacuum UV CD bands, indicating that the sequence can play a role in the vacuum UV CD of single-stranded RNA. The vacuum UV CD bands of the double-stranded (G.C)-containing RNAs, poly[r(G).r(C)] and poly[r(G-C).r(G-C)], were larger than the measured or estimated vacuum UV CD bands of their constituent single-stranded RNAs and were similar in having an exceptionally large positive band at about 185 nm and negative bands near 176 and 209 nm. These similarities were enhanced in difference-CD spectra, obtained by subtracting the CD spectra of the single strands from the CD spectra of the corresponding double strands. The (A.U)-containing double-stranded RNAs poly[r(A).r(U)] and poly[r(A-U).r(A-U)] were similar only in that their vacuum UV CD spectra had a large positive band at 177 nm. The spectrum of poly[r(A).r(U)] had a shoulder at 188 nm and a negative band at 206 nm, whereas the spectrum of poly[r(A-U).r(A-U)] had a positive band at 201 nm. On the other hand, difference spectra of both of the (A.U)-containing polymers had positive bands at about 177 and 201 nm. Thus, the difference-CD spectra revealed CD bands characteristic of A.U and G.C base pairing. (ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Raman spectroscopy of Z-form poly[d(A-T)].poly[d(A-T)

Helical structures of double-stranded poly[d(A-T)] in solution have been studied by Raman spectroscopy. While the classical right-handed conformation B-type spectra are obtained in the case of sodium chloride solutions, a Z-form Raman spectrum is observed by addition of nickel ions at high sodium concentration, conditions in which the inversion of the circular dichroic spectrum of poly[d(A-T)] is detected, similar to that observed for high-salt poly[d(G-C)] solutions [Bourtayre, P., Liquier, J., Pizzorni, L., & Taillandier, E. (1987) J. Biomol. Struct. Dyn. 5, 97-104]. The characterization of the Z-form spectrum of poly[d(A-T)] is proposed by comparison with previously obtained characteristic Raman lines of Z-form poly[d(G-C)] and poly[d(A-C)].poly[d(G-T)] solutions and of d(CG)3 and d(CGCATGCG) crystals [Thamann, T. J., Lord, R. C., Wang, A. H.-J., & Rich, A. (1981) Nucleic Acids Res. 9, 5443-5457; Benevides, J. M., Wang, A. H.-J., van der Marel, G. A., van Boom, J. H., Rich, A., & Thomas, G. J., Jr. (1984) Nucleic Acids Res. 14, 5913-5925]. Detailed spectroscopic data are presented reflecting the reorientation of the purine-deoxyribose entities (C2'-endo/anti----C3'-endo/syn), the modification of the phosphodiester chain, and the adenosine lines in the 1300-cm-1 region. The role played by the hydrated nickel ions in the B----Z transition is discussed.     

Anomalous excitonic CD of meso-tetrakis(3-N-methylpyridiniumyl)porphyrin bound to poly[d(A-T)(2)

When meso-tetrakis(3-N-methylpyridiniumyl)porphyrin (m-TMPyP) formed a complex with poly[d(A-T)(2)], an intense bisignate excitonic CD in the Soret absorption region was observed. The excitonic CD of the m-TMPyP-poly[d(A-T)(2)] complex is unique in that no other combination of the related porphyrin, namely, meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (where n = 2, 4), and polynucleotide including calf thymus DNA, poly[d(G-C)(2)], poly[d(I-C)(2)], and poly(dA).poly(dT), exhibits a comparable CD spectrum. From the [drug]/[DNA] ratio-dependence of the intensity and the shape of the CD spectrum, this porphyrin species is assigned to an extensively aggregated form. The extensively aggregated porphyrin disperses in 1 h after mixing to form moderately stacked porphyrin at a low mixing ratio. The magnitude of linear dichroism of the extensively aggregated porphyrin was small and the sign was negative in the Soret band, which indicated that the molecular plane of porphyrin in the complex is strongly tilted. On the other hand, the molecular plane of porphyrin is almost parallel to the DNA base plane (perpendicular to the DNA helix axis) in the moderately stacked form.     

CD of homopolymer DNA-RNA hybrid duplexes and triplexes containing A-T or A-U base pairs.

CD spectra and difference-CD spectra of (a) two DNA X RNA hybrid duplexes (poly[r(A) X d(U)] and poly[r(A) X d(T)]) and (b) three hybrid triplexes (poly-[d(T) X r(A) X d(T)], poly[r(U) X d(A) X r(U)], and poly[r(T) X d(A) X r(T)]) were obtained and compared with CD spectra of six A X U- and A X T-containing duplex and triplex RNAs and DNAs. We found that the CD spectra of the homopolymer duplexes above 260 nm were correlated with the type of base pair present (A-U or A-T) and could be interpreted as the sum of the CD contributions of the single strands plus a contribution due to base pairing. The spectra of the duplexes below 235 nm were related to the polypurine strands present (poly-[r(A)] or poly[d(A)]). We interpret the CD intensity in the intermediate 255-235 nm region of these spectra to be mainly due to stacking of the constituent polypurine strands. Three of the five hybrids (poly[r(A) X d(U)], poly[r(A) X d(T)], and poly[d(T) X r(A) X d(T)]) were found to have heteronomous conformations, while poly[r(U) X d(A) X r(U)] was found to be the most A-like and poly[r(T) X d(A) X r(T)], the least A-like.     

CD Spectral Comparisons of the Acid-Induced Structures of Poly[d(A)]Poly[r(A)], Poly[d(C)], and Poly[r(C)]

Abstract

CD spectra were used to compare the acid-induced structural transitions of poly[d(A)] and poly[d(C)] with those of poly[r(A)] and poly[r(C)], respectively. The types of base pairing were probably the same in the acid self-complexes of both A-containing polymers and in the acid self-complexes of both C-containing polymers. Similar base pairings were indicated by similarities in the difference CD spectra showing the changes during the first major acid- induced transitions of the polymers. Information from the CD spectra and pK_a values of the transitions suggested that the transitions for the RNA polymers involved similar structural changes. The two DNA polymers were markedly different. Single-stranded poly [d(A)] was in the most stacked structure and had the lowest pK^a for forming an acid self-complex. Single-stranded poly[d(C)] was in the least stacked structure and had the highest pK_a for forming a protonated duplex.     

Rotation of periphery methylpyridine of meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (n = 2, 3, 4) and its selective binding to native and synthetic DNAs

By utilizing circular and linear dichroism, the binding mode of meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (n = 2, 3, 4) to various DNAs was studied in this work. 2-N-(methylpyridiniumyl)porphyrin(o-TMPyP), in which rotation of the periphery pyridinium ring is prevented, exhibits similar spectral properties when bound to DNA, poly[d(G-C)(2)] and poly[d(A-T)(2)], suggesting a similar binding mode. Close analysis of the spectral properties led us to conclude that o-TMPyP sits in the major groove. However, both 3-N- and 4-N-(methylpyridiniumyl)porphyrin (m- and p-TMPyP), of which the periphery pyridinium ring is free to rotate, intercalate between the basepairs of DNA and poly[d(G-C)(2)]. In the presence of poly[d(A-T)(2)], m-TMPyP exhibits a typical bisignate excitonic CD spectrum in the Soret band, while p-TMPyP shows two positive CD bands. The excitonic CD spectrum of the m-TMPyP-poly[d(A-T)(2)] complex and the positive CD band of the o-TMPyP-poly[d(A-T)(2)] complex were not affected by the presence of the minor groove binding drug, 4',6-diamidino-2-phenylindole (DAPI), indicating that this porphyrin is bound in the major groove. In contrast, two positive CD bands of the p-TMPyP-poly[d(A-T)(2)] complex altered in the presence of DAPI. From the changes in CD spectrum and other spectral properties, a few possible binding modes for p-TMPyP to poly[d(A-T)(2)] are suggested.     

Stopped-flow kinetic analysis of the interaction of anthraquinone anticancer drugs with calf thymus DNA, poly[d(G-C)].poly[d(G-C)], and poly[d(A-T)].poly[d(A-T)]

The sodium dodecyl sulfate driven dissociation reactions of daunorubicin (1), mitoxantrone (2), ametantrone (3), and a related anthraquinone without hydroxyl groups on the ring or side chain (4) from calf thymus DNA, poly[d(G-C)]2, and poly[d(A-T)]2 have been investigated by stopped-flow kinetic methods. All four compounds exhibit biphasic dissociation reactions from their DNA complexes. Daunorubicin and mitoxantrone have similar dissociation rate constants that are lower than those for ametantrone and 4. The effect of temperature and ionic strength on both rate constants for each compound is similar. An analysis of the effects of salt on the two rate constants for daunorubicin and mitoxantrone suggests that both of these compounds bind to DNA through a mechanism that involves formation of an initial outside complex followed by intercalation. The daunorubicin dissociation results from both poly[d(G-C)]2 and poly[d(A-T)]2 can be fitted with a single exponential function, and the rate constants are quite close. The ametantrone and 4 polymer dissociation results can also be fitted with single exponential curves, but with these compounds the dissociation rate constants for the poly[d(G-C)]2 complexes are approximately 10 times lower than for the poly[d(A-T)]2 complexes. Mitoxantrone also has a much slower dissociation rate from poly[d(G-C)]2 than from poly[d(A-T)]2, but its dissociation from both polymers exhibits biphasic kinetics. Possible reasons for the biphasic behavior with the polymers, which is unique to mitoxantrone, are selective binding and dissociation from the alternating polymer intercalation sites and/or dual binding modes of the intercalator with both side chains in the same groove or with one side chain in each groove.     

Nuclear proteins from Drosophila melanogaster specifically binding to simple homopolymeric sequences of poly[d(T-G)].poly[d(C-A)] type

Binding of simple homopolymeric sequences to Drosophila melanogaster nuclear proteins has been studied. Proteins with Mr 65-72 kDa have been found, which specifically bind to synthetic poly[d(T-G)].poly[d(C-A)], as well as to D. melanogaster DNA containing a block of poly[d(T-G)].poly[d(C-A) 40 b.p. in length. It has been shown, that these proteins bind only to poly[d(T-G).poly[d(C-A)] and not to other types of simple sequences, for example poly[d(G-A)].poly[d(T-C)] and poly[d(A-T)].     

fd gene 5 protein binds to double-stranded polydeoxyribonucleotides poly(dA.dT) and poly[d(A-T).d(A-T)]

Circular dichroism (CD) data indicated that fd gene 5 protein (G5P) formed complexes with double-stranded poly(dA.dT) and poly[d(A-T).d(A-T)]. CD spectra of both polymers at wavelengths above 255 nm were altered upon protein binding. These spectral changes differed from those caused by strand separation. In addition, the tyrosyl 228-nm CD band of G5P decreased more than 65% upon binding of the protein to these double-stranded polymers. This reduction was significantly greater than that observed for binding to single-stranded poly(dA), poly(dT), and poly[d(A-T)] but was similar to that observed for binding of the protein to double-stranded RNA [Gray, C.W., Page, G.A., & Gray, D.M. (1984) J. Mol. Biol. 175, 553-559]. The decrease in melting temperature caused by the protein was twice as great for poly[d(A-T).d(A-T)] as for poly(dA.dT) in 5 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7. Upon heat denaturation of the poly(dA.dT)-G5P complex, CD spectra showed that single-stranded poly(dA) and poly(dT) formed complexes with the protein. The binding of gene 5 protein lowered the melting temperature of poly(dA.dT) by 10 degrees C in 5 mM Tris-HCl, pH 7, but after reducing the binding to the double-stranded form of the polymer by the addition of 0.1 M Na+, the melting temperature was lowered by approximately 30 degrees C. Since increasing the salt concentration decreases the affinity of G5P for the poly(dA) and poly(dT) single strands and increases the stability of the double-stranded polymer, the ability of the gene 5 protein to destabilize poly(dA.dT) appeared to be significantly affected by its binding to the double-stranded form of the polymer.