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 high specificity of incorporation of nucleotides into DNA by polymerase enzymes is crucial for maintaining fidelity of information transfer in cellular replication. The initial insertion event is the first point at which mutations of the genome are avoided. 1,2 Mismatched pairing at this step occurs on the level of only ∼1 in 10 3 -10 5 insertions, indicating a selectivity of at least 4 kcal/mol. 1 It is clear that polymerases enhance the selectivity of nucleotide choice at the active site relative to the much lower pairing differences observed at the duplex terminus in the absence of enzyme. 2 While many of the kinetic details of replication have been studied in recent years, 3 the precise physical origins of this selectivity enhancement are poorly understood. Mechanisms involving both kinetic and binding selectivity between correct and incorrect nucleotides have been proposed. 3 Base-base hydrogen bonding, base stacking, base pair geometry, and interactions between the enzyme, DNA, and nucleotides have all been invoked as potentially important interactions; however, the relative importance of these different effects remains unclear. Many DNA nucleotide analogs with altered or reduced H-bonding potential have been examined as substrates for polymerases; 4 most of those analogs are quite poor substrates, and result in less discriminate incorporation fidelity than do the natural nucleotides. This general finding has been used as evidence that the number and strength of hydrogen bonds in a given pair determine efficiency and fidelity of DNA synthesis. Indeed, most if not all current models for replication fidelity hold that the specificity of hydrogen bonds formed in the new base pair is a central contributor to the observed selectivity.Here we present evidence, however, that a DNA polymerase can exert high fidelity even when a base pair completely lacks conventional hydrogen bonds. The difluorotoluene nucleoside 1 has recently been constructed as a nonpolar shape mimic for natural thymidine (2). 5,6 Its "base" moiety cannot measurably form paired complexes with adenine derivatives even in chloroform, a solvent in which H-bonded complexes are much more stable than they are in water. 7 When placed within a DNA strand paired opposite adenine, moreover, it actually destabilizes the helix by ∼4-5 kcal relative to thymine at the same position. In addition, 1 shows no inherent pairing selectivity among the four natural bases, also consistent with its nonpolar, nonhydrogen-bonding nature (ref 7 and work in progress). We felt therefore that 1 would serve as a good test for the importance of thymidine's hydrogen bonding groups on fidelity, because 1 lacks the strongly localized charges but retains nearly the exact steric shape of the natural molecule. If, as current models suggest, such polar interactions are important for achieving high fidelity, then 1 would be expected to be very inefficient and highly nonselective as a template for replication.© 1997 American Chemical Society * Author to whom correspondence should...

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo ؊ mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (V max ͞K m ) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F-A pair is associated with a brief pause, whereas that beyond A in the inverted A-F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A-F͞F-A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide͞ template shape complementarity may play a more important role than previously believed.

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...

This paper reviews progress in the study of Tibetan Plateau (TP) climate dynamics over the past decade. Several theoretical frameworks, including thermal adaptation and the TP sensible heat (SH) driving air-pump, have been developed to identify the mechanisms responsible for the circulation anomaly produced by thermal forcing of the TP. Numerical simulations demonstrate that the thermal effects of large-scale orography, including the Tibetan and Iranian Plateaus (TIP), are crucial for the formation of the East Asian and South Asian summer monsoons (SASM) because the surface SH of the TIP is the major driver of the water vapor transport required for the genesis of the north branch of the SASM. The large-scale orography of the TP affects the Asian climate through thermal forcing in spring and summer, and mechanical forcing in winter. The TP forcing can also influence the Asian summer monsoon (ASM) onset over the Bay of Bengal (BOB) by enhancing the BOB warm pool at the surface and by modulating the South Asian High (SAH) in the upper troposphere. On intra-seasonal timescales, the TP thermal forcing significantly modulates spring rainfall in southern China and generates the biweekly oscillation of the SAH in summer. Despite climate warming, the atmospheric heat source over the TP, particularly the spring SH, exhibits a clear weakening trend from the 1980s to 2000s. This weakening of the spring SH contributed to the anomalous ‘dry in the north’ and ‘wet in the south’ rainfall pattern observed over East China. Also discussed are challenges to further understanding the mechanism of TP forcing on the multi-scale variability of the ASM.