DNA ADDUCT FORMATION BY PLATINUM ANTICANCER DRUGS - INSIGHT INTO AN UNUSUAL GPG INTRASTRAND CROSS-LINK IN A HAIRPIN-LIKE DNA OLIGONUCLEOTIDE USING NMR AND DISTANCE GEOMETRY METHODS

Treatment of a self-complementary duplex with Pt anticancer drugs leads to formation of an unusual type of hairpin-like oligonucleotide, Pt(A(2)){5'd(A(1)T(2)G(3)G(4)*G(5)*T(6)A(7)C(8)C(9)C(10)A(11)T(12))3'}(A(2) = cis-(NH3)(2) or ethylenediamine (en) and G*'s are platinated at N7). In previous NMR studies, several residues exhibited abnormal upfield- and downfield-shifted (1)Hand P-31 signals, and one aromatic H-1 signal, G(4)*H8, could not be located. In the present study, we found the G(4)*H8 signal to be broad and shifted similar to 2 ppm upfield into the H1' spectral region. This shift change is much large; than the theoretical maximum (<1 ppm) predicted for an unstrained structure, suggesting the platination site is strained. Distance geometry (DG) structures were calculated from NMR data in order to elucidate the structural basis for the unique properties of these species, including the unusual NMR shifts. At the platination site, the Pt(en) moiety is located in the major groove, and the platinum-bound G's both possess some unusual features; G(4)* has an N sugar conformation and G(5)* has a syn conformation. The syn G(5)* base is oriented perpendicular to the Pt coordination plane. In contrast, the G(4)* base is almost coplanar with this plane; the unusual orientation forces G(4)*H8 into a close (less than or equal to 3 Angstrom) clash with the five-membered ring of G(5)*. This proximity explains the substantial upfield shift observed for G(4)*H8. The broadness of the G(4)*H8 signal could be explained by minor vacillations about the Pt-N7(4) bond, which would sweep G(4)*H8 across different shielding regions of the anisotropic G(5)* base. The A(7) base in Do models is tucked inside the hairpin loop, with A(7)H8 close to G(5)*H8. A(7) is correctly oriented to explain (a) the G(4)*H2' shift into the methyl region of the H-1 spectrum, (b) the strong A(7)H8 to G(5)*H8 NOE, and (c) the downfield-shifted A(7)H8 signal (caused by G(5)* deshielding). The unprecedented downfield P-31 shift of A(7)pC(8) is a result of an anti,anti conformation about the zeta,alpha torsion angles induced by a distortion in the backbone needed to allow G(4)*C-9 Watson-Crick base pairing at the top of the stem. A B-DNA-like helical stem was found with base pairing between the first four bases of the 3' and 5' ends (A(1)T(12), T(2)A(11), G(3)C(10), and G(4)*C-9). These features are supported by 2D NOESY-in-H2O data. Such hairpin-like structures, induced by the need to balance DNA and Pt structural demands, could form in palindromic regions of DNA and could be instrumental in platinum drug activity.