Hydrophobic, Non-Hydrogen-Bonding Bases and Base Pairs in DNA

Abstract

We report the properties of hydrophobic isosteres of pyrimidines and purines in synthetic DNA duplexes. Phenyl nucleosides 1 and 2 are nonpolar isosteres of the natural thymidine nucleoside, and indole nucleoside 3 is an analog of the complementary purine 2-aminodeoxyadenosine. The nucleosides were incorporated into synthetic oligodeoxynucleotides and were paired against each other and against the natural bases. Thermal denaturation experiments were used to measure the stabilities of the duplexes at neutral pH. It is found that the hydrophobic base analogs are nonselective in pairing with the four natural bases but selective for pairing with each other rather than with the natural bases. For example, compound 2 selectively pairs with itself rather than with A, T, G, or C; the magnitude of this selectivity is found to be 6.5-9.3 °C in Tm or 1.5-1.8 kcal/mol in free energy (25 °C). All possible hydrophobic pairing combinations of 1, 2, and 3 were examined. Results show that the pairing affinity depends on the nature of the pairs and on position in the duplex. The highest affinity pairs are found to be the 1-1 and 2-2 self-pairs and the 1-2 heteropair. The best stabilization occurs when the pairs are placed at the ends of duplexes rather than internally; the internal pairs may be destabilized by imperfect steric mimicry which leads to non-ideal duplex structure. In some cases the hydrophobic pairs are significantly stabilizing to the DNA duplex; for example, when situated at the end of a duplex, the 1-1 pair is more stabilizing than a T-A pair. When situated internally, the affinity of the 1-1 pair is the same as, or slightly better than, the analogous T-T mismatch pair, which is known to have two hydrogen bonds. The studies raise the possibility that hydrogen bonds may not always be required for the formation of stable duplex DNA-like structure. In addition, the results point out the importance of solvation and desolvation in natural base pairing, and lend new support to the idea that hydrogen bonds in DNA may be more important for specificity of pairing than for affinity. Finally, the study raises the possibility of using these or related base pairs to expand the genetic code beyond the natural A-T and G-C pairs.

Figure 1

The structures of hydrophobic nucleosides 1–3. The nucleoside “bases” are abbreviated F (difluorotoluene), B (trimethylbenzene), and D (dimethylindole).

Figure 2

The structures of the T–A and T–T pairs in DNA and proposed structures for the analogous hydrophobic purine-pyrimidine and pyrimidine–pyrimidine pairs.

Figure 3

Proton NMR titration of 9-ethyladenine with l-cyclohexy- luracil (●)and with nucleoside l a (■). Experiments were carried out in CDCl₃ at 25 °C with 1 mM 9-ethyladenine, following the N resonance on 9-ethyladenine.

Figure 4

The pairing of hydrophobic nucleosides 1–3 with the natural bases and with themselves in the center of a 12-base pair duplex, as measured by thermal melting temperature (see Table 1 for conditions).

Figure 5

Pairing of hydrophobic nucleosides 1–3 with themselves and each other in self-complementary duplex, as compared to natural T–A and T–T pairs. All duplexes contain the same core sequence d(CG)_3, and are substituted externally or internally with the pairs shown See Table 3 for conditions and complete sequences.

Figure 6

Illustrations of three different pairing situations seen in this study (A–C). It is proposed that the first (A, natural H-bonded pairing) and third (C, hydrophobic pairing) are more favorable because there are no uncompensated desolvations in base pair formation. The second (B, hydrophobic–hydrophilic pairing) is disfavored by ~5 kcal/mol relative to the first because of energetically costly desolvation of the natural base.