Efforts toward the expansion of the genetic alphabet: Information storage and replication with unnatural hydrophobic base pairs

The faithful recognition of the interstrand hydrogen bonds between compleme ntary nucleobases forms the foundation of the genetic code. The ability to replicate DNA containing a stable third base pair would allow for an expans ion of the information content of DNA by supplementing the existing two bas e pairs of the genetic alphabet with a third. We report the optimization of unnatural nucleobases whose pairing in duplex DNA is based on interbase hy drophobic interactions. We show that the stability and selectivity of such unnatural base pairs may be comparable to, or even exceed, that of native p airs. We also demonstrate that several unnatural base pairs are incorporate d into DNA by Klenow fragment of Escherichia coli DNA polymerase I with an efficiency equivalent to that of native DNA synthesis. Moreover, the unnatu ral bases are orthogonal to the native bases, with correct pairing being fa vored by at least an order of magnitude relative to mispairing.