Polymerase Evolution: Efforts toward Expansion of the Genetic Code

摘要

The biological system of information storage, based on the selective Watson-Crick hydrogen bonding (H-bonding) of adenine with thymine (dA:dT base pair) and guanine with cytosine (dG:dC base pair), has been conserved throughout nature. Expansion of the alphabet to contain a third base pair would allow additional information to be encoded in DNA and would also enable a variety of in vitro experiments using unnatural nucleic acids. A priori, there is no reason to assume that the requirements for duplex stability and replication must limit the genetic alphabet to only two base pairs, or even to hydrogen-bonded base pairs. The stability and replication of DNA containing nucleobase analogues that are predominantly hydrophobic and that do not bear a H-bonding pattern or shape that is complementary to the natural bases have been previously examined. One class of unnatural base pairs that has been extensively examined is that formed between two identical nucleobase analogues. For example, the PICS self-pair is stable in duplex DNA and synthesized by the Klenow fragment of E. coli (Kf) and by Thermus aquaticus (Taq) polymerase, but not by the proteolytic fragment of Taq (Stoffel fragment, Sf). In addition, none of these polymerases can extend a primer terminating with a PICS self-pair.