The high resolution IH and 31P NMR spectra of the (dG-dC)8 duplex have been recorded in low-and high-salt solutions in order to evaluate the structural aspects of the saltinduced transition of oligo (dG-dC) (2) demonstrated that the circular dichroism of oligo(dG-dC) and poly(dG-dC) inverts in the presence of a high salt concentration. This reversible transition exhibits an activation energy of 22 kcals in both directions (2). The conformational transition was also observed when 60% (final concentration) ethanol (3) or the antibiotic mitomycin was added to poly(dG-dC) in aqueous solution (4). The trypanocidal agent ethidium bromide binds to the low-salt form of poly(dG-dC) more strongly than to the high-salt form (5). The salt-induced transition was not observed for the synthetic RNA poly(G-C) or the synthetic DNAs poly(dA-dT) and poly(dG)-poly(dC) (2). These results suggest that conformational features related to helical type, base composition, and base sequence play a crucial role in governing the presence or absence of the structural transition.Drew et al. (6) have observed that crystals of dC-dG-dC-dG grown in low-and high-salt solutions exhibit different space groups, and they demonstrated a reversible transition between the two crystalline states.We have monitored the 'H and 31p NMR parameters for (dG-dC)n as a function of salt concentration in an attempt to elucidate the structural aspects of the salt-induced conformational transition. Because the NMR line widths are narrower at the oligomer level, this paper discusses the NMR chemical shifts for oligo(dG-dC) of chain lengths 16 and 20-30. EXPERIMENTALThe isolation and characterization of (dG-dC)n oligomers of chain lengths 16 (n = 8) and 20-30 (n = 10-15) have been reported (7). They were passed through Sephadex GlO columns prior to the recording of NMR spectra.High-resolution 360-MHz proton NMR spectra were recorded in the continuous wave (CW) mode for the exchange- (Fig. 1). The base protons can be readily assigned, whereas it is not yet possible to definitively differentiate among protons on the sugar ring linked to the guanosine and cytidine residues. The cytidine H-5 and H-6 protons, which shift upfield on duplex formation, are sensitive indicators of base pair overlap geometries in the duplex state. By contrast, the sugar H-i' chemical shifts predominantly monitor changes in the glycosidic torsion angles associated with the duplex-tostrand transition.The magnitude and direction of the chemical shift changes associated with the duplex-to-strand transition of (dG-dC)8 (Fig. 1) and poly(dG-dC) are similar to values reported for the melting transition of poly(dA-dU) (8), poly(dA-dT) (8), and poly(dI-dC) (9). These alternating purine-pyrimidine polynucleotide duplexes adopt B-DNA type overlap geometries in solutions (8, 10).Salt-Induced (dG-dC)8 Transition. Circular dichroism spectra have been recorded for oligo(dG-dC) duplexes as a function of salt concentration. The extent'of the structural transition for (dG-dC)8 as a function of NaCl is...

The helix-coil transition of the octanucleotide self-complementary duplex dG-dG-d A-dA-dT-dTdC-dC has been monitored at the Watson-Crick protons, the base and sugar nonexchangeable protons and the backbone phosphates by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The melting transition of the octanucleotide monitored by ultraviolet absorbance spectroscopy is characterized by the thermodynamic parameters AH" = -216.7 kJ/mol and AS" (25 "C) = -0.632 kJ mol-' K-' in 0.1 M NaCl, 10 mM phosphate solution. Correlation of the transition midpoint values monitored by the ultraviolet absorbance studies at strand concentrations below 0.2 mM and by NMR studies at 5.3 mM suggest that both methods are monitoring the octanucleotide duplex-to-strand transition. The NMR spectra of the Watson-Crick ring NH protons of the octanucleotide duplex have been followed as a function of temperature. The resonance from the terminal dG-dC base pairs broadens out at room temperature while the resonances from the other base pairs broaden simultaneously with the onset of the melting transition. The nonexchangeable base and sugar H-1' protons are resolved in the duplex and strand states and shift as average peaks through the melting transition. The experimental shifts on duplex formation have been compared with calculated values based on ring-current and atomic diamagnetic anisotropy contributions for a B-DNA base-pair-overlap geometry in solution. Several nonexchangeable proton resonances broaden in the fast-exchange region during the duplex-to-strand transition and the excess widths yield a duplex dissociation rate constant for the octanucleotide of 1.9 x lo3 s-' at 32 "C (fraction of duplex = 0.86) in 0.1 M NaCl, 10 mM phosphate buffer. The 31P resonances of the seven internucleotide phosphates are distributed over 0.6 ppm in the duplex state, shift downfield during the duplex-to-strand transition and undergo additional downfield shifts during the stacked-to-unstacked strand transition with increasing temperature.The application of nuclear magnetic resonance (NMR) spectroscopy to monitor the duplex-to-strand transition of oligonucleotide and polynucleotide sequences has been recently reviewed and demonstrates the power of this technique to differentiate between individual base pairs in a duplex and, in addition, to monitor each base pair at the Watson-Crick hydrogen bonds, the nonexchangeable base and sugar protons and the backbone phosphates in solution [1,2].These investigations include studies on the exchangeable and nonexchangeable protons of the deoxy pentanucleotide (dT-dT-dG-dT-dT) . (dA-dA-dC-dAdA) [3,4] and deoxy block polymer (dC,,-dA,,) . (dT,,-dG,,) [5], and the self-complementary sequences dA-dT-dG-dC-dA-dT [6] and poly(dA-dT) [7]. In addition, the exchangeable protons in double-stranded and triple-stranded oligomer duplexes containing Watson-Crick and Hoogsteen base pair have been

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The well-resolved base and sugar proton resonances in the high resolution proton nuclear magnetic resonance (NMR) spectra of poly (dA-dT) Arnott and coworkers (1) have recently extended the early studies of Davies and Baldwin (2) on the x-ray diffraction patterns from oriented, crystalline fibers of poly(dA-dT). They propose a novel, 8-fold helical structure, with an axial rise per residue of 3.03 A and a C3 exo sugar pucker conformation. Baldwin and coworkers (3-6) have investigated the helix-coil transition of poly(dA-dT) and circular oligo(dAdT). They have characterized the role of hairpin and straight chain helices by monitoring absorbance melting curves as a function of salt, temperature, and pH. The thermodynamic parameters for the helix-coil transition of poly(dA-dT) have been elucidated from differential heat calorimetry (7,8).High resolution nuclear magnetic resonance (NMR) spectroscopy is capable of monitoring many aspects of the helixcoil transition of alternating deoxypurine-deoxypyrimidine oligonucleotides in aqueous solution. These studies include investigations of the exchangeable and nonexchangeable protons in the duplex of the self-complementary hexanucleotide d(A-T-G-C-A-T) (9-12) and the d[C-G(-C-G)n], n = 1,2 duplexes (13) in aqueous solution.The Watson-Crick thymine imino protons in the oligomer duplexes d(pT-A)n, n = 3,4,5, were monitored in our laboratory as a function of temperature by high resolution proton NMR spectroscopy in H20 solution (14). The chemical shifts and line widths of these exchangeable resonances were obAbbreviation: NMR, nuclear magnetic resonance. servable when the double helix was predominantly intact but broadened with increasing coil concentration. An alternate NMR approach monitors the entire helix-coil transition as followed by the nonexchangeable protons and the, phosphates in D20 solution (11)(12)(13)(15)(16)(17).Nuclear magnetic resonance spectral line widths yield kinetic information about the helix-coil transition when the exchange rate is on the order of the chemical shift difference between the helix and coil states. It will be demonstrated that the adenine H2 proton in poly(dA-dT) exhibits an about 1 ppm chemical shift difference between the two states and provides a handle for monitoring the kinetic aspects of the helix-coil transition. EXPERIMENTAL Sample preparationThe alternating copolymer poly(dA-dT) samples for proton NMR studies were purchased from Long Island Biochemical Corp. and were shipped in Tris buffer (chain length: about 300 nucleotides).270 MHz Proton NMR Studies. The purchased sample in Tris buffer was lyophilized and the spectra were recorded on 18.8 mM (with respect to phosphorus) poly (dA-dT)

The helix-to-coil transition of the synthetic DNA poly(dA-dT) in the presence of the anthracycline antitumor antibiotic daunomycin has been investigated by high-resolution proton nuclear magnetic resonance (NMR) spectroscopy in 1 M salt solution. The dissociation of the complex, containing molar ratios of phosphate to daunomycin (Pi/drug) of 50, 25, 9 and 5, with increasing temperature can be monitored independently at the nucleic acid and the antibiotic resonances under conditions of fast exchange. The antibiotic complex formation shifts suggest that either ring B and/or C of the intercalated anthracycline chromophore of daunomycin overlaps with adjacent nucleic acid base pairs. Ultraviolet/visible melting studies of daunomycin complexes with a series of synthetic DNAs substituted with halogen atoms (Br, I) at position 5 of the pyrimidine ring suggest that intercalation of the antibiotic into poly(dA-dU) is not perturbed by bulky substituents at this position. A comparison of the melting curves for the daunomycin . poly(dA-dT) complex with an analog of the antibiotic where the NH3+ group is replaced by dimethylglycine demonstrates the important contributions of electrostatic interactions between the amino sugar and backbone phosphates to the stability of the complex in low salt solution. The ultraviolet/visible and NMR studies monitor biphasic melting transitions at the nucleic acid markers in the daunomycin . poly(dA-dT) complexes, Pi/drug = 50 -9, so that antibiotic-free base-pair regions and those centered about bound daunomycin can be independently studied at the synthetic DNA level in solution.There has been considerable interest in the elucidation of the structural and kinetic aspects of complexes formed between antibiotics, mutagens and carcinogens with nucleic acids in solution. These studies include the application of ultraviolet/visible, circular dichroism and fluorescence spectroscopy, as well as, viscosity and sedimentation investigations [l -71. High-resolution NMR spectroscopy is a powerful method for investigating the structure of macromolecules in solution [8 -131 and we present below an example of the interaction of an antitumor agent with a synthetic DNA in aqueous solution.Daunomycin (Scheme 1) belongs to the class of glycosidic anthracycline antibiotics (which includes adriamycin and carminomycin) which have been successfully used in combination chemotherapy against acute leukemia and solid tumors in man 114,151. It appears that the dose-dependent cardiac toxicity of the anthracyclines may be minimized by the addition Abbreviations. NMR, nuclear magnetic resonance; P,/drug, molar ratio of phosphate in poly(dA-dT) to daunomycin, brsdU, 5-bromo-deoxyuridine ; ?dU, 5-iodo-deoxyuridine. The present study attempts to elucidate structural features of the intercalated complex of daunomycin and nucleic acids by high-resolution NMR spectro-