Factors contributing to aromatic stacking in water: Evaluation in the context of DNA

We report the use of thermodynamic measurements in a self-complementary DNA duplex (5'-dXCGCGCG)(2), where X is an unpaired natural or nonnatural deox ynucleoside, to study the forces that stabilize aqueous aromatic stacking i n the context of DNA. Thermal denaturation experiments show that the core d uplex (lacking X) is formed with a free energy (37 degrees C) of -8.1 kcal mol(-1) in a pH 7.0 buffer containing 1 M Na+ We studied the effects of add ing single dangling nucleosides (X) where the aromatic "base" is adenine, g uanine, thymine, cytosine, pyrrole, benzene, 4-methylindole, 5-nitroindole, trimethylbenzene, difluorotoluene, naphthalene, phenanthrene, and pyrene. Adding these dangling residues is found to stabilize the duplex by an addit ional -0.8 to -3.4 kcal mol(-1). At 5 mu M DNA concentration, T-m values ra nge from 41.7 degrees C (core sequence) to 64.1 degrees C (with dangling py rene residues). For the four natural bases, the order of stacking ability i s A > G greater than or equal to T = C. The nonpolar analogues stack more s trongly in general than the more polar natural bases. The stacking geometry was confirmed in two cases (X = adenine and pyrene) by 2-D NOESY experimen ts. Also studied is the effect of ethanol cosolvent on the stacking of natu ral bases and pyrene. Stacking abilities were compared to calculated values for hydrophobicity, dipole moment, polarizability, and surface area. In ge neral, hydrophobic effects are found to be larger than other effects stabil izing stacking (electrostatic effects, dispersion forces); however, the nat ural DNA bases are found to be less dependent on hydrophobic effects than a re the more nonpolar compounds. The results also point out strategies for t he design nucleoside analogues that stack considerably more strongly than t he natural bases; such compounds may be useful in stabilizing designed DNA structures and complexes.