CYTOSINE CYTOSINE+ BASE-PAIRING STABILIZES DNA QUADRUPLEXES AND CYTOSINE METHYLATION GREATLY ENHANCES THE EFFECT

Previous spectroscopic studies demonstrated that the oligodeoxynucleotide d(CGC G3 GCG) undergoes a reversible cation-dependent transition between Watson-Crick (WC) hairpin and parallel-stranded '' ''G-DNA'' quadruplex structures [Hardin, C. C., Watson, T., Corregan, M., & Bailey, C. (1992) Biochemistry 31, 833-841]. The relative stabilities of the structures were assessed as a function of pH, and it was found that the quadruplex was substantially stabilized (DELTAT(m) = +15-degrees) when the pH was shifted from 7.5 to 6 (apparent pK(a) = 6.8). In the present study, the effects of different cations and pH on four specific sequence variants were determined to test the proposal that this stabilization is due to C.C+ base pair formation mediated by N3-protonation of cytosine. Characteristically large differences in stability were observed when structures formed by d(TAT G3 ATA) and d(TAT G4 ATA) were thermally dissociated at pH 7 in the presence of different cations, verifying that G(n) tracts bordered by TAT- and -ATA sequences form quadruplex structures. Imino proton NMR results indicate that the d(m5C G m5C G3 G m5C G)4 and d(TAT G4 ATA)4 quadruplex structures are parallel-stranded. It was necessary to increase the K+ concentration from 40 mM to ca. 200 mM to stabilize d(TAT G3 ATA)4, while the d(TAT G4 ATA)4 complex was nearly as stable as the quadruplex formed by d(CGC G3 GCG) under the same conditions. The d(TAT G4 ATA)4 quadruplex was only slightly stabilized at pH 6 relative to pH 7.5 (DELTAT(m) = +3-degrees-C), confirming that the unique stabilization that occurs in the pH 6.8 range with [d(CGC G(n) GCG)4.ion(n)] complexes is due to the C residues. The sequence d(M5C G m5C G3 G m5C G) was found to form a very stable quadruplex in K+ or Ca2+. As with the quadruplex formed by the unmethylated analog, the stability is greatly enhanced when the pH is decreased below about 7.2 (pK(a,obs) = 6.8). Dissociation kinetic constants and activation energies were determined for quadruplexes formed by d(CGC G3 GCG), d(m5C G m5C G3 G M5C G) and d(TAT G4 ATA). Quantitative comparisons showed that methylation produces a complex that is much more stable at pH 7 in 40 mM Na+ than either of the unmodified structures; the rate-limiting activation energy for dissociation of d(CGC G3 GCG)4 was 22 kcal mol-1 less than for the methylated analog. Statistical analysis of the kinetic data showed that at least three distinct processes occurred with the C- and m5C-containing molecules, while only two different processes could be resolved with the d(TAT G4 ATA) quadruplex. The results suggest that cytosine methylation stabilizes the complex primarily by producing more favorable entropic (stacking) interactions, not by causing a positive shift in the pK(a) for cytosine protonation. To summarize, the following factors can act alone or synergistically to stabilize DNA quadruplexes: (1) moderate increases in the concentrations of K+ or Ca2+, and to a smaller degree Na+, within the physiological ranges, (2) moderate pH decreases through the ranges that occur in eukaryotic nuclei, and (3) methylation of cytosine residues at GCG sites.