Proton and phosphorus-31 NMR investigations of actinomycin D binding selectivity with oligodeoxyribonucleotides containing multiple adjacent d(GC) sites

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.

Drug—DNA sequence‐dependent interactions analysed by electric linear dichroism

“…The phenoxazinone chromophore of actinomycin is not parallel to DNA base pairs. It is well established that full intercalation of the chromophore is precluded on steric grounds because of the bulky cyclic peptides attached (Chen, 1988;Scott et al, 1988;Zhou et al, 1989). Previous ELD measurements (Hogan et al, 1979) confirmed a lower degree of intercalation of actinomycin D compared to ethidium bromide, proflavine or 9-aminoacridine.…”

Binding of actinomycin D to DNA: evidence for a nonclassical high-affinity binding mode that does not require GpC sites.

“…Inspection of the differential melting curves in Fig. 3 (7,14,(47)(48)(49)(50) as well as differences in drug-drug and drug-DNA interactions when two ActD molecules bind to adjacent rather than nonadjacent sites (18,19). In this connection, the x-ray studies of Berman and coworkers (47,49) should be noted.…”

“…In this connection, ActD has been shown to exhibit a binding preference for the 3' side ofguanine residues (10,11). The dinucleotide site GpC exhibits an especially high binding affinity for ActD (11,14,15,18,19,21). Other dinucleotide sites, such as GpT, GpA, GpG, and even CpG, also have been proposed as potential ActD binding sites, although most of the supporting evidence has been indirect and sometimes contradictory (9,(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

“…1) is attributed to its ability to inhibit transcription by binding to double-stranded DNA. For this reason, the DNA binding properties of ActD have been studied extensively for many years (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). In general, these binding studies have shown that ActD complexes with duplex DNA by intercalation of its planar, aromatic rings between adjacent base pairs of the double helix.…”

Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting

“…Secondly when potential actinomycin binding sites are in close proximity (for example in GCGC) only one of the potential sites can be occupied at a time. Although some NMR studies have shown that it is possible to bind two actinomycin molecules to the tetranucleotide GCGC [29], binding of the second molecule is highly anticooperative and it is generally believed that the exclusion site size for actinomycin, under normal conditions, is at least 4 base pairs [1]. Sites [2][3][4] Nonetheless it is possible to make some observations concerning the relative dissociation rates from some of the sites.…”