We report the use of thermodynamic measurements in a self-complementary DNA duplex (5′-dXCGCGCG) 2 , where X is an unpaired natural or nonnatural deoxynucleoside, to study the forces that stabilize aqueous aromatic stacking in the context of DNA. Thermal denaturation experiments show that the core duplex (lacking X) is formed with a free energy (37 °C) of −8.1 kcal·mol −1 in a pH 7.0 buffer containing 1 M Na + . We studied the effects of adding single dangling nucleosides (X) where the aromatic "base" is adenine, guanine, 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 additional −0.8 to −3.4 kcal·mol −1 . At 5 μM DNA concentration, T m values range from 41.7 °C (core sequence) to 64.1 °C (with dangling pyrene residues). For the four natural bases, the order of stacking ability is A > G ≥ T = C. The nonpolar analogues stack more strongly in general than the more polar natural bases. The stacking geometry was confirmed in two cases (X = adenine and pyrene) by 2-D NOESY experiments. Also studied is the effect of ethanol cosolvent on the stacking of natural bases and pyrene. Stacking abilities were compared to calculated values for hydrophobicity, dipole moment, polarizability, and surface area. In general, hydrophobic effects are found to be larger than other effects stabilizing stacking (electrostatic effects, dispersion forces); however, the natural DNA bases are found to be less dependent on hydrophobic effects than are the more nonpolar compounds. The results also point out strategies for the design nucleoside analogues that stack considerably more strongly than the natural bases; such compounds may be useful in stabilizing designed DNA structures and complexes.
The phosphorylation of IkappaB by the IKK complex targets it for degradation and releases NF-kappaB for translocation into the nucleus to initiate the inflammatory response, cell proliferation, or cell differentiation. The IKK complex is composed of the catalytic IKKalpha/beta kinases and a regulatory protein, NF-kappaB essential modulator (NEMO; IKKgamma). NEMO associates with the unphosphorylated IKK kinase C termini and activates the IKK complex's catalytic activity. However, detailed structural information about the NEMO/IKK interaction is lacking. In this study, we have identified the minimal requirements for NEMO and IKK kinase association using a variety of biophysical techniques and have solved two crystal structures of the minimal NEMO/IKK kinase associating domains. We demonstrate that the NEMO core domain is a dimer that binds two IKK fragments and identify energetic hot spots that can be exploited to inhibit IKK complex formation with a therapeutic agent.
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...
The physical and chemical factors that allow DNA to perform its functions in the cell have been studied for several decades. Recent advances in the synthesis and manipulation of DNA have allowed this field to move ahead especially rapidly during the past fifteen years. One of the most common chemical approaches to the study of interactions involving DNA has been the use of DNA base analogues in which functional groups are added, deleted, blocked, or rearranged. Here we describe a different strategy, in which the polar natural DNA bases are replaced by nonpolar aromatic molecules of the same size and shape. This allows the evaluation of polar interactions (such as hydrogen bonding) with little or no interference from steric effects. We have used these nonpolar nucleoside isosteres as probes of noncovalent interactions such as DNA base pairing and protein - DNA recognition. We have found that, while base-pairing selectivity does depend on Watson - Crick hydrogen bonding in the natural pairs, it is possible to design new bases that pair selectively and stably in the absence of hydrogen bonds. In addition, studies have been carried out with DNA polymerase enzymes to investigate the importance of Watson - Crick hydrogen bonding in enzymatic DNA replication. Surprisingly, this hydrogen bonding is not necessary for efficient enzymatic synthesis of a base pair, and significant levels of selectivity can arise from steric effects alone. These results may have significant impact both on the study of basic biomolecular interactions and in the design of new, functionally active biomolecules.
Conventional microprocessors use elementary logic gates to perform complex computational tasks. Mimicking such computational processes using purely molecular systems has been limited in most cases by the lack of design generality or potential addressability of existing molecular logic gates. Herein we report that by employing the universal recognition properties of DNA simple photonic logic gates can be created that are capable of AND, NAND, and INHIBIT logic operations.
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