No abstract

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with adjacent 5'-GC-3' sites. ActD showed very high specificity for binding to GC sites regardless of oligomer length and surrounding sequence. For a first class of duplexes with a central GCGC sequence, a mixture of 1:1 complexes was observed due to the two different orientations of the ActD phenoxazone ring system. Analysis of 1H chemical shifts suggested that the favored 1:1 complex had the benzenoid side of the phenoxazone ring over the G base in the central base pair of the GCGC sequence. This is the first case in which an unsymmetrical intercalator has been shown to bind to DNA in both possible orientations. A unique 2:1 complex, with significantly different 1H and 31P chemical shifts relative to those of the 1:1 complexes, was formed with these same oligomers, again with the benzenoid side of the ActD molecule over the G base of the central GC base pair. There is considerable anticooperativity to binding of the second ActD in a GCGC sequence. In titrations of oligomers with the GCGC sequence, only the two 1:1 complexes are found up to ratios of one ActD per oligomer. Increasing the ActD concentration, however, resulted in stoichiometric formation of the unique 2:1 adduct. Spectrophotometric binding studies indicated that the apparent binding equilibrium constant for a GC site adjacent to a bound site is reduced by approximately a factor of 20 relative to the ActD binding constant to an isolated GC site.

Dimerization of coralyne, Ia, and its propyl analogue Ic has been analyzed at neutral pH as a function of ionic strength. Even at low ionic strength (I = 0.02) dimerization constants (molar units) for both compounds are 10(5) or greater, which is considerably larger than values obtained for similar intercalating molecules. Coralyne seems to undergo association to higher aggregates somewhat easier than Ic, which could be due to the fact that the propyl group on Ic provides some steric hinderance in forming higher aggregates. Both compounds readily associate with DNA. At high ratios of ligand to DNA, the spectra for Ia or Ic are similar to that of a highly aggregated complex. At low ratios of ligand to DNA, the spectra for both compounds approach a limit, which is relatively independent of ionic strength and concentration, and can be identified with the intercalated species. The slight differences between Ia and Ic in dimerization or in association with DNA do not seem capable of explaining the significant differences in antileukemic activity of these compounds.

To evaluate the length and sequence dependence of the unusual interaction properties observed for nonalternating A/T sequences in deoxyribonucleic acid (DNA) [Wilson, W. D., Wang, Y. H., Krishnamoorthy, C. R., & Smith, J. C. (1985) Biochemistry 24, 3991-3999], we have synthesized the oligomers d(A-T)6, dA10 X dT10, and d(A6-T6) and evaluated their interaction with the intercalator propidium. Propidium visible spectral shifts on adding all three oligomers are quite similar. Low-temperature spectrophotometric binding measurements indicate that d(A-T)6 has a significantly larger binding constant for propidium than dA10.dT10, as with the analogous alternating and nonalternating DNA polymers. The oligomer dA10.dT10 displays positive cooperativity in its propidium binding isotherm, and its binding constant increases with increasing temperature while d(A-T)6 does not display positive cooperativity, and its binding constant decreases with temperature, again as with the analogous polymers. van't Hoff plots indicate that the propidium binding enthalpies are approximately -9 and +6 kcal/mol for the alternating and nonalternating DNA samples, respectively. The mixed-sequence self-complementary oligomer d(A6-T6) has an unusual low-temperature binding isotherm which suggests a single strong binding site and a larger number of weaker binding sites which bind propidium cooperatively. A van't Hoff plot indicates that the cooperative sites d(A-T)6 have binding constants and binding enthalpies similar to dA10.dT10. Similar rate constants are observed in the sodium dodecyl sulfate driven dissociation reaction of propidium from d(A-T)6 and d(A6-T6), but the association reaction of propidium is significantly slower with d(A6-T6) than with d(A-T)6.(ABSTRACT TRUNCATED AT 250 WORDS)

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with one GC binding site [d(ATATGCATAT) (1), d-(ATACGCGTAT) (2), and d(ATATACGCGTATAT) (3)] and with two GC sites [d(ATGCATGCAT) (4)]. At R = 1 (molar ratio of ActD to oligomer duplex) ActD caused a doubling of the number of imino proton signals at, and adjacent to, the GC binding site of 1. One of the G.C base pair signals shifted upfield while the other shifted downfield. Both of the signals for the A.T base pairs adjacent to the binding site shifted downfield. All imino proton signals of 2 and the longer sequence, 3, shifted upfield on binding of ActD to the GC site, indicating a sequence-dependent change in base stacking on complex formation. For both 1 and 2 addition of ActD resulted in a similar pattern of three downfield 31P NMR signals. The two most downfield signals have chemical shift and temperature dependence which are characteristic of phosphate groups at isolated intercalation sites. At R = 1 the ActD complex with 4 has very complex spectra with both upfield and downfield A.T and G.C imino signals. All these data were consistent with two 1:1 complexes with the unsymmetrical phenoxazone ring adopting both of the two possible orientations.(ABSTRACT TRUNCATED AT 250 WORDS)