NMR Solution Structure of a DNA Decamer Containing an Interstrand Cross-link of the Antitumor Drugcis-diamminedichloroplatinum(II)

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Abstract
A 10 base pairs double-stranded oligonucleotide with the sequence d(CCTCG*CTCTC). d(GAGAG*CGAGG) containing a single interstrand cross-link resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(G*C/G*C) site by a cis-diammineplatinum(II) residue was analyzed by 1H NMR spectroscopy. All the exchangeable and nonexchangeable protons resonance lines (except some H5′-H5″) were assigned. NOESY spectra and chemical shifts indicated that the cross-linkage of the guanines of G*5 and G*6 induced extrahelicity of C5 and C6. Moreover, several unusual proximities were observed such as: (i) NOE cross-peaks between the H2′-H2″ of G*5 or G*6 and the aromatic proton of their 5′ neighbor C4 or A7 (ii) the absence of cross-peak for the steps G*5-C6, C6-T7 and C5-G4 (iii) a strong NOESY connectivity between H8(G*5) and H2(A7). All these data allowed us to describe the head to tail arrangement of the two cross- linked guanines as well as their stacking with flanking neighbor nucleotides (G*5 with T7.A7 base pair and G*6 with C4.G4 base pair). Using all the NOESY and TOCSY data (208 constraints), we have obtained a solution structure of the cross-linked duplex by using the NMR-constrained molecular mechanics program JUMNA. The reversal position of the two cross-linked guanines placed the cis-diammineplatinum(II) residue in the minor groove. The stacking of the two cross-linked guanines with the surrounding bases induced a bend of 40° toward the minor groove. The locally left-helix formation, the extrusion of the cytosines and the stacking of the platinated guanines led to an unwinding of 76°. This value is in good agreement with the values deduced from gel electrophoresis experiments.