Proton and phosphorus nmr studies of d‐CpG(pCpG)_n duplexes in solution. Helix–coil transition and complex formation with actinomycin‐D

Abstract

SynopsisThe Watson-Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG),, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range Oo-90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes a t low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located a t the end of the helix from those in an interior position. There is fraying a t the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson-Crick ring imino protons exchange a t a faster rate than the Watson-Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG), double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix-coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 X sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D20, pH 6.25 a t 27OC. The well-resolved 31P resonances for the internucleotide phosphates of the tetramer and hexamer sequences a t superconducting fields shift upfield by 0.2-0.5 pprn on helix formation. These shifts reflect a conformational change about the w,w' phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG), helix-coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by -2.6 ppm and -1.6 pprn on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs a t the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain a t the 0-P-0 backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG), duplexes exhibit a B-DNA conformation in solution. 533

Interaction of platinum compounds with short oligodeoxynucleotides containing guanine and cytosine

“…These values are indicative of the presence of one platinum atom/ oligonucleotide molecule, assuming a slight decrease of the cmax value upon binding of cisPt [lo, 141. At the reaction temperature used a large proportion of the selfcomplementary oligodeoxynucleotides are in the doublestranded form [20,211. Therefore, the above-mentioned observations show that cisPt has a strong preference for intrastrand cross-linking.…”

Nuclear magnetic resonance studies of the helix-coil transition of poly (dA-dT) in aqueous solution.

“…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.…”

“…The 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).…”

Steroid diamine-nucleic acid interactions: partial insertion of dipyrandium between unstacked base pairs of the poly(dA-dT) duplex in solution.

“…The steroid diamine is nonplanar and therefore unable to insert into such a site while spanning the backbone phosphates through its charged ends. It therefore appears that the steroid diamine must partially insert into a binding site generated by unstacked (21), and complexation shifts of t2 ppm are observed for actinomycin D-nucleic acid interactions (22). The (Figs.…”