Efforts toward Expansion of the Genetic Alphabet: DNA Polymerase Recognition of a Highly Stable, Self-Pairing Hydrophobic Base

Abstract

The storage and replication of biological information is based on the specific hydrogen-bonding patterns of the Watson-Crick A:T and G:C base pairs.1 The generation of enzymatically replicable unnatural base pairs with thermal stability comparable to that of A:T and G:C would significantly expand the biological and chemical potential of DNA. One strategy for expanding the genetic alphabet has focused on the synthesis of bases with altered hydrogen-bonding patterns.2-8 However, in light of the dominant role played by hydrophobicity in protein folding and stability,9-11 we have explored the use of hydrophobic and van der Waals interactions to generate an orthogonal base pair for the storage and replication of genetic information. Previous experiments in which hydrophobic bases were incorporated into duplex DNA led to a destabilization of the duplex relative to the native pairs.12-18 In the course of systematically evaluating a variety of predominantly hydrophobic nucleobases,19 we have characterized a base, which cannot form H-bonds, whose self-pair stabilizes duplex DNA relative to an A:T or G:C base pair. The 7-propynyl isocarbostyril nucleoside (1) was synthesized and converted into the nucleoside triphosphate (4) and phosphoramidite (5) as shown in Scheme 1. When incorporated into duplex DNA, the 1:1 interstrand interaction is expected to be completely hydrophobic in the absence of significant backbone deformation; the keto groups are designed to be in the minor groove and can H-bond to solvent. The thermal stability of the base pairs was evaluated by determining the melting temperature (Tm) of the duplex 7 with X,Y ) G:C, A:T, or 1:1 (Table 1). The 1:1 hydrophobic base pair stabilizes the DNA duplex relative to