Experimental Measurement of Aromatic Stacking Affinities in the Context of Duplex DNA

Abstract

Noncovalent interactions between aromatic molecules are widely believed to be important contributing factors in the stabilization of organized structure in biological macromolecules. 1,2 Among the most significant aromatic-aromatic interactions are those found in helical nucleic acid structures. Since the identity of the nearest neighbors to a given base pair is the best single predictor of thermodynamics in DNA duplexes, 3 it is clear that aromatic π-π interactions are crucial to the stabilization of these structures. 4 While there have been a considerable number of theoretical studies aimed at modeling the π-π interaction in DNA, 5 there have been remarkably few experimental studies specifically addressing the thermodynamics of stacking (separate from base pairing) in DNA itself. 6 For that reason we have undertaken a study of aromatic stacking in the context of duplex DNA, and we hope to begin to elucidate what are the important forces which stabilize this organized structure. We report here the first experimental comparison of the stacking abilities of natural DNA bases and of nonnatural aromatic analogs in double-stranded DNA.To separate stacking from pairing (hydrogen-bonding) interactions in duplex DNA we placed the natural or nonnatural nucleotide of interest in a "dangling" position (without a pairing partner) at the end of a base-paired duplex (Figure 1). 7 The resulting stabilization of the duplex by the dangling base can be measured by thermal denaturation experiments, with comparison to the duplex lacking the added nucleotide.Electrostatic effects resulting from such localized charge have been implicated both in the stabilization and in the geometry of aromatic stacking. 5 To examine such effects we compared not only natural DNA bases but also nonpolar molecules with similar shape and surface area. Thus, we compared the DNA base thymine (1) and adenine (3) with their respective nonpolar isosteres difluorotoluene (2) and 4-methylindole (4). 9 We also compared the stacking of the aromatic hydrocarbons benzene (5), naphthalene (6), phenanthrene (7), and pyrene (8). The synthesis of these nucleoside analogs has been reported. [10][11][12][13] Results of the thermodynamic measurements made at pH 7.0 and 1 M NaCl are presented in Table 1 Supporting Information Available:Plots of thermodynamic data, sample thermal melting profiles, and proton NMR spectra (3 pages). See any current masthead page for ordering and Internet access instructions. Measurement of the duplexes with dangling thymine and adenine residues shows, perhaps not surprisingly, that the purine stacks on the duplex more strongly than the smaller pyrimidine base. The two unpaired deoxyadenosines add 2.0 kcal of stabilizing interaction to the selfcomplementary sequence, and thymines add 1.1 kcal to the duplex stability. This relative stacking ability is as predicted from nearest-neighbor parameters 3 and is consistent with dangling-end studies carried out in RNA. 7 Interestingly, the data show that the nonpolar DNA base mimics stack cons...

A Uniform Mechanism Correlating Dangling-End Stabilization and Stacking Geometry

“…The solvation energy of the dangling base will also drive the stacking equilibrium toward the stacked form, thus directly affecting the dynamics of the dangling end. This is in agreement with the recently published results that have shown that the higher the hydrophobicity and polarizability (a m ) of the dangling end, the more stability it provides to the duplex when attached as dangling end (37,42), even if the stacking areas and geometries are presumably constant. We also propose that the reason that further stabilization is gained when additional dangling bases are added to an overhang sequence is that the additional dangling nucleotides reduce the dynamics of the innermost dangling base by nearest-neighbor intrastrand stacking interactions.…”

Solid‐State NMR Studies on Supramolecular Chemistry

“…In the last two decades π-stacking interactions between aromatic systems have been extensively studied. This interaction has been proposed to be a pivotal assembly force in many important supramolecular systems including various protein and enzyme substrates, 53 nucleic acids, 54 and molecular catalysts. 55 A deeper understanding of the nature of the interaction is the key step for developing the synthesis of controlled nanoscale structures.…”

Factors Contributing to Aromatic Stacking in Water: Evaluation in the Context of DNA

“…A previous preliminary study reported dangling end measurements for a few of these analogues but did not include all four natural bases, did not include structural data, and did not correlate the results with physical properties or solvent effects. 50 …”