The DNA oligonucleotide d(CGCG3GCG) can form either a Watson-Crick (WC) hairpin or a parallel-stranded quadruplex structure containing six G-quartet base pair assemblies. The exchange between these forms and single strands can be monitored using circular dichroism (CD). NMR results verified the assignment of specific CD bands to quadruplex and hairpin species, respectively. Cations stabilize the quadruplex in the order K+ greater than Ca2+ greater than Na+ greater than Mg2+ greater than Li+ and K+ greater than Rb+ greater than Cs+, indicating that K+ has an optimum ionic radius for complex formation and that ionic charge affects the extent of ion-induced stabilization. The quadruplex is stable in the presence of 40 mM K+ at micromolar DNA concentration and can be kinetically trapped as a metastable form when prepared at millimolar DNA concentration and then diluted into buffer containing 40 mM Na+. The concentration of K+ required to reverse the equilibrium from the hairpin to the quadruplex decreases sharply with increased DNA concentration. The quadruplex has an unusual pKa of ca. 6.8, indicating that C.C+ base pairs are probably forming. This system provides insights into some of the detailed structural characteristics of a ["G4-DNA".ion] complex and an experimental model for the recently proposed "sodium-potassium conformational switch" [Sen, D., & Gilbert, W. (1988) Nature 334, 364-366; Sen, D., & Gilbert, W. (1990) Nature 344, 410-414]. These results may help to explain the lack of cytidine residues in G-rich telomeric DNAs and suggest that methylation of GC-rich duplex DNAs in "GpC islands" may induce quadruplex formation within heterochromatin domains, resulting in reversible chromosomal condensation.

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 (delta Tm = +15 degrees C) when the pH was shifted from 7.5 to 6 (apparent pKa = 6.8). In the present study, the effects of different cations and pH on four specific sequence varients 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 Gn 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 (delta Tm = +3 degrees C), confirming that the unique stabilization that occurs in the pH 6.8 range with [d(CGC Gn GCG)4.ionn] 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 (pKa,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.(ABSTRACT TRUNCATED AT 400 WORDS)

The series of cooperative transitions that lead to [d(TG4)4.(K+)m] quadruplex assembly upon rapid addition of KCl to d(TG4) strands were studied. Quadruplex samples were dialyzed against KCl then Li-EDTA and found to retain between three and five strongly bound potassiums with affinities >10(6) M-2. Absorbance thermal denaturation (melt) and circular dichroism (CD) equilibrium binding data were obtained. The latter were analyzed using two classes of binding models to simulate the effects of the assumed intermolecular interactions on the binding curves (isotherms). The melt experiments yielded equilibrium dissociation constants (Kd) ranging from 10(-11) to 10(-12) M3 at the melting temperatures. Extrapolating these values to 23 degrees C predicts Kd values in the 10(-28) M3 range if the heat capacity (Cp) is not strongly dependent upon temperature changes over this range. Assuming Ka is equal to 1/Kd (from melting analyses), very large association free energies stabilize the quadruplex at 23 degrees C in 100 mM KCl (DeltaGa = -43 kcal mol-1). Plots of the differential melt curve peak half-widths, a measure of cooperativity, versus d(TG4) concentration showed that quadruplex dissociation is much more cooperative at 400 mM KCl than at 100 mM KCl. Forty-eight hour quadruplex assembly time courses were monitored by CD at 264 nm. Equilibrium quadruplex accumulation generally required over 10 h, and net reaction extents were in the 10-85% range. Hill plots of the data show that initial steps in the multistep pathway are positively cooperative, presumably due to strong strand-cation and strand-strand binding interactions in duplex and triplex assembly reactions, then negatively cooperative in quadruplex formation. Models were developed to rationalize the experimental observations in terms of consecutive cooperative allosteric transitions from cation-deficient relaxed (R) strand-aggregates to cation-containing tense (T) structures, driven by the allosteric effector K+. Quantitative mappings of positive and then negative cooperativity were obtained by fitting the results as a function of strand number incorporated during quadruplex assembly. Surprisingly, models for reactions involving incorporation of five and six strands fit the data better than models involving only four strands. The 5-step "induced fit" model fits the data as well as or better than 3- and 4-step models and better than all of the strand aggregation models that were devised and investigated. Net association free energies (summation operatori=1,n) ranged from -20 to -26 kcal mol-1, approximately half the magnitude of the apparent stabilities measured by absorbance melts. Likely explanations for this discrepancy involve hysteresis and errors due to inadequate equilibration in the melt experiments. Hysteresis is thought to be produced by irreversibility due to different predominant mechanisms in absorbance (dissociation) and CD (association) experiments. The kinetic block to quadruplex assembly can be unambiguously attributed to quadruplex formation and not...

In 10 mM sodium phosphate, pH 7.6, containing 0.1 mM ethylenediaminetetraacetic acid, ions correspondings to the non-calent, four-stranded oligonucleotide, d(CGCG4GCG)4, were detected by negative ion electrospray ionization (ESI) mass spectrometry at a low nozzle-skimmer (delta NS) bias (-150 V), but not at a higher delta NS bias (> -250 V). In contrast, when the sample was desalted and analyzed by ESI mass spectrometry at a low delta NS bias only ions for the single-stranded d(CGCG4GCG) species were observed. These data agree with spectroscopic evidence which showed that oligonucleotides with the sequence motif 5'd(CGCGnGCG)3', where n = 2-5, formed stable four-stranded complexes in the presence of monatomic cations, like K+, Ca2+, Na+ and Li+, but not in their absence.