Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

A monoclonal antibody to triplex DNA binds to eucaryotic chromosomes.

A monoclonal antibody (Jel 318) was produced by immunizing mice with poly[d(TmC)].poly[d(GA)].poly[d(mCT) which forms a stable triplex at neutral pH. Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs such as poly[d(TG)].poly[d(CA)]. In addition the antibody did not recognize pyrimidine.purine DNAs containing mA (e.g. poly[d(TC)].poly[d(GmA)]) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing. The binding of Jel 318 to chromosomes was assessed by immunofluorescent microscopy of mouse myeloma cells which had been fixed in methanol/acetic acid. An antibody specific for duplex DNA (Jel 239) served as a control. The fluorescence due to Jel 318 was much weaker than that of Jel 239 but binding to metaphase chromosomes and interphase nuclei was observed. The staining by Jel 318 was unaffected by addition of E. coli DNA but it was obliterated in the presence of triplex. Since an acid pH favours triplex formation, nuclei were also prepared from mouse melanoma cells by fixation in cold acetone. Again Jel 318 showed weak but consistent staining of the nuclei. Therefore it seems likely that triplexes are an inherent feature of the structure of eucaryotic DNA.     

The alternating conformation of analogues of poly[d(AT)].

Synthetic DNAs were prepared containing 6-methyl adenine (m6A) in place of adenine and 5-ethyl uracil (Et5U) or 5-methoxymethyl uracil (Mm5U) in place of thymine. All three modifications destabilized duplex DNAs to varying degrees. The binding of ethidium was studied to analogues of poly[d(AT)]. There was no evidence of cooperative binding and the "neighbour exclusion rule" was obeyed in all cases although the binding constant to poly[d(m6AT)] was approximately 6 fold higher than to poly[d(AT)]. 31P NMR spectra were recorded in increasing concentrations of CsF. Poly[d(AEt5U)] showed two well-resolved signals separated by 0.55 ppm in 1 M CsF compared to 0.32 ppm for poly[d(AT)] under identical conditions. In contrast, poly[d(AMm5U)] and poly[d(m6AT)] showed two signals separated by 0.28 ppm and 0.15 ppm respectively, only when the concentration of CsF was raised to 2 M. The signals for poly[d(AT)] in 2 M CsF were better resolved and were separated by 0.41 ppm. These results suggest that minor modifications to the bases may have conformational effects which could be recognized by DNA-binding proteins.     

Structure of the alpha-form of poly[d(A)].poly[d(T)] and related polynucleotide duplexes

The alpha-form of poly[d(A)].poly[d(T)], observed in fibers at high (greater than 80%) relative humidity, is a 10-fold double-helical structure of pitch 3.2 nm. This new X-ray analysis shows that the two strands of the double helix are of the same kind conformationally and both B-like in containing C-2'-endo-puckered deoxyribose rings. Nevertheless, the two strands are different enough for the overall morphology of the duplex to resemble that of the heteromerous model for the drier (beta) form of poly[d(A)].poly[d(T)] in which one strand has C-2'-endo rings and the other C-3'-endo. Since the orientations of the bases in poly[d(A)].poly[d(T)] are persistently different from those of classical B-DNA it is likely that there will be local bending (about 10 degrees) at the junctions between general sequence tracts and the oligo[d(A)].oligo[d(T)] tracts that occur in some native DNAs. The conclusions about the structure of alpha-poly[d(A)].poly[d(T)] are reinforced by independent analyses of similar X-ray diffraction patterns from poly[d(A)].poly[d(U)] and poly[d(A-I)].poly[d(C-T)].     

Ethidium bromide does not fluoresce when intercalated adjacent to 7-deazaguanine in duplex DNA.

Several synthetic DNAs were prepared containing the unusual bases 7-deazaadenine (c7A) and 7-deazaguanine (c7G). As judged from changes in melting temperatures these modified DNAs bound ethidium to a similar extent as the parent polymers. However, duplexes such as poly [d(Tc7G)].poly[d(CA)] and poly[d-(TC)].poly[d(c7GA]) gave no enhancement of ethidium fluorescence in a standard ethidium fluorescence assay. Fluorescence spectra in the range 400-650 nm showed that ethidium bound to poly[d(TC)].poly[d(Gc7A)] gave 70% of the fluorescence of the parent polymer poly[d(TC)].poly[d(GA)], whereas the fluorescence of poly[d(TC)].poly[d(c7GA)] was essentially 0%. Even the intrinsic fluorescence of ethidium in solution was quenched in the presence of poly[d(TC)].poly[d(c7GA)]. Binding constants were estimated from Scatchard analysis and were 4.8, 3.4, and 2.0 x 10(6) M-1 for poly[d(TC)].poly[d(GA)], poly[d(TC)].poly[d(Gc7A)], and poly[d(TC)].poly[d(c7GA)], respectively. This reduction in binding constant cannot account for the loss of fluorescence. The UV spectrum of ethidium was measured in the presence of these DNAs, and some significant differences were noted. Presumably the presence of 7-deazaguanine alters the electronic structure of bound ethidium so that it can no longer fluoresce.     

CD evidence that the alternating purine-pyrimidine sequence poly[d(A-C).d(G-T)], but not poly[d(A-T).d(A-T)], undergoes an acid-induced transition to a modified secondary conformation.

Circular dichroism and UV absorption data showed that poly[d(A-C).d(G-T)] (at 0.01M Na+ (phosphate), 20 degrees C) underwent two reversible conformational transitions upon lowering of the pH. The first transition was complete at about pH 3.9 and resulted in an acid form of the polymer that was most likely a modified, protonated duplex. The second transition occurred between pH 3.9 and 3.4 and consisted of the denaturation of this protonated duplex to the single strands. UV absorption and CD data also showed that the separated poly[d(A-C)] strand formed two acid-induced self-complexes with pKa values of 6.1 and 4.7 (at 0.01M Na+). However, neither one of these poly[d(A-C)] self-complexes was part of the acid-induced rearrangements of the duplex poly[d(A-C).d(G-T)]. Acid titration of the separated poly[d(G-T)] strand, under similar conditions, did not show the formation of any protonated poly[d(G-T)] self-complexes. In contrast to poly[d(A-C).d(G-T)], poly[d(A-T).d(A-T)] underwent only one acid-induced transition, which consisted of the denaturation of the duplex to the single strands, as the pH was lowered from 7 to 3.     

Poly(pyrimidine) . poly(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH

Poly(pyrimidine) . poly(purine) tracts have been discovered in the 5'-flanking regions of many eucaryotic genes. They may be involved in the regulation of expression since they can be mapped to the nuclease-sensitive sites of active chromatin. We have found that poly(pyrimidine) . poly(purine) DNAs which contain 5-methylcytosine (e.g. poly[d(Tm5C)] . poly[d(GA)]) will form a triplex at a pH below 8. In contrast, the unmethylated analogue, poly[d(TC)] . poly[d(GA)] only forms a triplex at pHs below 6. Synthetic DNAs containing repeating trinucleotides and poly[d(Um5C)] . poly[d(GA)] behave in a similar manner. Thus the stability of a triplex can be controlled by methylation of cytosine. This suggests a model for the regulation of expression based upon specific triplex formation on the 5'-side of eucaryotic genes.     

Synthetic repeating sequence DNAs containing phosphorothioates: nuclease sensitivity and triplex formation.

More than twenty repeating sequence DNAs containing phosphorothioates were prepared from the appropriate dXTPs with DNA polymerase I. The Tms of the modified DNAs were all lower than the parent polymers. A phosphorothioate group 5' to a pyrimidine gave rise to a large decrease than 5' to a purine, e.g., poly(dA).poly(dT) = 50 degrees; poly(dsA).poly(dT) = 44 degrees; poly(dA).poly(dsT) = 33 degrees; and poly(dsA).poly(dsT) = 26 degrees. The presence of phosphorothioate groups had a dramatic effect on triplex formation; poly[d(TC)].poly[d(sGsA)] spontaneously dismutases to a triplex at pH 8 whereas triplex formation in poly[d(sTsC)].poly[d(GA)] was inhibited. Surprisingly poly(dsG).poly(dC) had a Tm which initially decreased with increasing ionic strength. Resistance to digestion with pancreatic DNAse I did not correlate with phosphorothioate content. Poly[d(AsT)], poly[d(TsC)].poly[d(sGA)] and poly[d(sTG)].poly[d(sCA)] were resistant whereas poly[d(sAT)] and poly[d(sTsTG)].poly[d(CsAsA)] were rapidly degraded. Thus phosphorothioate groups cause small conformational changes and may reveal new families of conformational polymorphisms.     

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)].     

Temperature dependence of the volumetric parameters of drug binding to poly[d(A-T)].Poly[d(A-T)] and Poly(dA).Poly(dT)

We report the temperature and salt dependence of the volume change (DeltaVb) associated with the binding of ethidium bromide and netropsin with poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]. The DeltaV(b) of binding of ethidium with poly(dA).poly(dT) was much more negative at temperatures approximately 70 degrees C than at 25 degrees C, whereas the difference is much smaller in the case of binding with poly[d(A-T)].poly[d(A-T)]. We also determined the volume change of DNA-drug interaction by comparing the volume change of melting of DNA duplex and DNA-drug complex. The DNA-drug complexes display helix-coil transition temperatures (Tm several degrees above those of the unbound polymers, e.g., the Tm of the netropsin complex with poly(dA)poly(dT) is 106 degrees C. The results for the binding of ethidium with poly[d(A-T)].poly[d(A-T)] were accurately described by scaled particle theory. However, this analysis did not yield results consistent with our data for ethidium binding with poly(dA).poly(dT). We hypothesize that heat-induced changes in conformation and hydration of this polymer are responsible for this behavior. The volumetric properties of poly(dA).poly(dT) become similar to those of poly[d(A-T)].poly[d(A-T)] at higher temperatures.     

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.     

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)].     

Synthesis and characterization of poly[d(G-z5C)]. B-Z transition and inhibition of DNA methylase

Deoxy-5-azacytidine 5'-triphosphate was synthesized and used as a substrate for the enzymatic synthesis of the polynucleotide poly[d(G-z5C)]. Whereas the triphosphate decomposes in solution, the azacytosine analogue incorporated into DNA is stable under conditions preserving the double-helical structure. Poly[d(G-z5C)] undergoes the transition to the left-handed Z conformation at salt (NaCl and MgCl2) concentrations approximately 30% higher than those required for unsubstituted poly[d(G-C)]. However, the incorporation of azacytidine potentiates the formation at room temperature of the Z helix stabilized by the transition metal Mn2+; in the case of poly[d(G-C)], a heating step is required. The spectral properties of the two polymers in the B and Z forms are similar. Both left-handed forms are recognized by anti-Z DNA immunoglobulins, indicating that the DNAs bear common antigenic features. Poly[d(G-z5C)] is not a substrate for the DNA cytosine 5-methyltransferase from human placenta. It is a potent inhibitor of the enzyme when tested in a competitive binding assay. These results are compatible with a very strong, possibly covalent, mode of interaction between methyltransferases and DNA containing 5-azacytosine.     

Crystallization of immunoglobulin Fab fragments specific for DNA

The purification and crystallization of Fab fragments of two mouse monoclonal immunoglobulins specific for different DNA structures are described. In each case, papain digestion of the immunoglobulins produced a mixture of Fab species differing in their isoelectric points. Purification of one of these species was required to obtain suitable crystals. One of these antibodies, Jel 72, is specific for right-handed duplex poly(dG).poly(dC). An Fab fragment of Jel 72 with a pI of 8.8 was purified by anion-exchange chromatography and used to obtain crystals from 56% saturated ammonium sulfate and 50 mM sodium acetate, pH 4.2, that diffract to 2.6-A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 94.6, b = 102.6, c = 92.4 A. The other antibody, Jel 318, binds triple-stranded DNA poly[d(Tm5C)].poly[d(GA)].poly[d(m5C + T)]. Jel 318 Fab fragments with isoelectric points of 7.6 and 7.8 were also purified by anion-exchange chromatography, and crystals were obtained from 12% polyethylene glycol 8000, 50 mM NaCl, and 10 mM Tris.HCl, pH 7.8. These crystals diffract to about 2.4-A resolution and also belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 82.4, b = 139.5, and c = 42.0 A. For both Fab fragments, crystal size and quality improved dramatically upon purification of an individual isoelectric species.     

Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells.

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.     

Left-handed helical structure of poly[d(A-C)].poly[d(G-T)] studied by infrared spectroscopy

Infrared spectroscopic studies demonstrate the ability of poly[d(A-C)].poly[d(G-T)] to adopt a Z-type conformation. The Z form of the unmodified polynucleotide is induced by Ni2+ counterions and not by Na+. The B----Z equilibrium is shifted at room temperature, in the presence of 1 Ni2+/nucleotide, by an increase in the concentration of poly[d(A-C)].poly[d(G-T)]. The importance of specific binding of Ni2+ ions on the N7 site of purines in the stabilization of the Z form is also discussed.     

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.     

Bh-DNA: variations of the poly[d(A)].poly[d(T)] structure within the framework of the fibre diffraction studies

A refinement of the recent results for poly[d(A)].poly[d(T)] (Alexeev et al., J. Biomol. Struct. Dyn. 4,989 (1987)) involving additional parameters of the base-pair structure and of the sugar-phosphate backbone expands the conformational potential of this polynucleotide of the B type to include the possibility of bifurcated hydrogen bonds of the kind recently discovered in crystalline deoxyoligonucleotide with lone d(A)n.d(T)n stretch (Nelson et al., Nature 330, 221 (1987)). Still, analysis of the available data and energy calculations do not seem to indicate that the bifurcated H-bonds are a crucial factor responsible for the anomalous structure of the d(A)n.d(T)n sequence. The unique structural properties of poly[d(A)].poly[d(T)] can hardly be explained without taking into account its interactions with the double-layer hydration spine in the minor groove. In view of the hydration mechanism stabilizing poly[d(A)].poly[d(T)] and of the polynucleotide's heteronomous prehistory (Arnott et al., Nucleic Acids Res. 11,4141 (1983)) we suggest that this B-type structure be called Bh.     

Thermodynamic analysis of monoclonal antibody binding to duplex DNA.

A technique based on fluorescence polarization (anisotropy) was used to measure the binding of antibodies to DNA under a variety of conditions. Fluorescein-labeled duplexes of 20 bp in length were employed as the standard because they are stable even at low ionic strength yet sufficiently short so that both arms of an IgG cannot bind to the same duplex. IgG Jel 274 binds duplexes in preference to single-stranded DNA; in 80 mM NaCl Kobs for (dG)20.(dC)20 is 4.1x10(7) M-1 compared with 6.4x10(5) M-1 for d(A5C10A5). There is little sequence specificity, but the interaction is very dependent on ionic strength. From plots of log Kobs against log[Na+] it was deduced that five or six ion pairs are involved in complex formation. At low ionic strength,Kobs is independent of temperature and complex formation is entropy driven with DeltaH degrees obs and DeltaC degrees p,obs both zero. In contrast, in 80 mM NaCl DeltaC degrees p,obs is -630 and -580 cal mol-1K-1 for [d(TG)]10.[d(CA)]10 and (dG)20.(dC)20 respectively. IgG Jel 241 also binds more tightly to duplexes than single-stranded DNA, but sequence preferences were apparent. The values for Kobs to [d(AT)]20 and [d(GC)]20 are 2.7x10(8) and 1.3x10(8) M-1 respectively compared with 5.7x10(6) M-1 for both (dA)20. (dT)20 and (dG)20.(dC)20. As with Jel 274, the binding of Jel 241 is very dependent on ionic strength and four or five ionic bonds are involved in complex formation with all the duplex DNAs which were tested. DeltaC degrees p,obs for Jel 241 binding to [d(AT)]20 was negative (-87 cal mol-1K-1) in 80 mM NaCl but was zero at high ionic strength (130 mM NaCl). Therefore, for duplex-specific DNA binding antibodies DeltaC degrees p,obs is dependent on [Na+] and a large negative value does not correlate with sequence-specific interactions.     

CD of six different conformational rearrangements of poly[d(A-G).d(C-T)] induced by low pH.

On the basis of circular dichroism (CD) data, we have now identified six different conformational states (other than the duplex) of poly[d(A-G).d(C-T)] at pH values between 8 and 2.5 (at 0.01M Na+; 20 degrees C). Three of these structural rearrangements were observed as the pH was lowered from 8 to 2.5, and three additional rearrangements were observed as the pH was raised from 2.5 back to neutral pH. The major components of the six conformational states were defined using appropriate combinations of the CD spectra of the duplex, triplex, and denatured forms of this polymer, as well as the CD spectra of the individual single strands and their respective acid-induced self-complexes. Our results show that the acid-induced rearrangements of poly[d(A-G).d(C-T)] include not only the poly[d(C+-T).d(A-G).d(C-T)] triplex, but also include the poly[d(C-T)] loop-out structure and a self-complexed form of the poly[d(A-G)] strand that is pH-dependent.