Multinuclear NMR investigation of zinc(2+) binding to a dodecamer oligodeoxyribonucleotide: insights from carbon-13 NMR spectroscopy

Abstract

The IH and I3C NMR spectra of the self-complementary dodecamer d(AIT2G,G~G5T6A,CsC9CIOAIIT12)2 were assigned. The dodecamer chemical shifts and NOE effects suggest a normal B-form duplex at 12 OC. However, weak NOE cross peaks from the AH2 signal to the HI' signal of the 3' nucleotide were observed. These weak cross peaks are consistent with some minor sliding in of the propeller-twisted base pairs, particularly at the center of the duplex. At lower concentrations or at higher temperature, a second form of the oligonucelotide was evident. This concentration dependence, the downfield chemical shifts of some 'H resonances, and the observation of an imino signal at 11 ppm strongly imply that this minor form is a hairpin. Addition of Zn2+ greatly disfavored this hairpin form. At lower oligonucleotide concentration, Zn2+ was twice as effective as Mg2+ in favoring the duplex form. The imino 'H spectra established that GC base pairing is not disrupted in the duplex form, even at high levels of added Zn2+. At higher oligonucleotide concentration, when the duplex was converted to the random coil form by temperature elevation, no hairpin form was detected in the presence of Zn2+, whereas such a form was clearly present, particularly at 42 OC, in the absence of Zn2+. At low temperature, when Zn2+ was added, the H8 signals at G4 and, to a lesser extent, G, shifted downfield with little effect on the G5H8 or any AH8 signals. This result suggests a preferential binding to N7 of G4. This conclusion was strongly supported by downfield shifts of the G, and G , C8 signals and the absence of appreciable changes in the C8 signals of other purines. In addition, the C5 signals of G4 and G , shifted upfield and the C4 signals broadened. These characteristic changes strongly support coordination to N7 for these two G residues. However, no significant changes in NOE cross-peak intensities, the C3' I3C signals, or the ,lP NMR signals were observed. Therefore, it is clear that GN7-MCrN7 cross-linked or GMG sandwich species are not formed, since the strain in the sugar-phosphate backbone that would be produced by such species would have led to appreciable changes in chemical shift and NOE cross-peak intensity. The selectivity pattern (G, > G 3 > G5, A) is consistent with the expected molecular electrostatic potential on the basis of literature calculations. Thus, although the study of spectroscopically silent labile metal ions such as ZnZ+ is difficult, a multinuclear NMR investigation of the target biomolecule can be quite revealing.