We are interested in nucleic acids containing the four natural bases guanine, adenine, cytosine and thymine as well as two additional unnatural bases which form a stable pair between themselves, but not with any of the natural bases, and which can be replicated in vitro. We are exploring unnatural base pairs, which possess hydrogen binding arrangements that are different from the natural nucleobases or which are more hydrophobic. Several predominantly hydrophobic bases have been synthesized which were designed to be complementary to one another and not to the natural bases. We are systematically evaluating the differing factors that contribute to the stabilization of a base pair in duplex DNA versus those that stabilize a base pair in a polymerase active site during the enzymatic synthesis of DNA including hydrogen-bonding, base shape, hydrophobicity and polarizability. We will characterize the bases thermodynamically and structurally. To evolve a polymerase capable of synthesizing DNA containing three base pairs we describe a novel selection scheme based on activity. A library of polymerase mutants is first displayed on phage. Oligonucleotide, which may act as a substrate, is then used as a potential site of attachment to solid support. Desired polymerase activity results in the cleavage from, or the attachment to, the solid support. The phage particle may then be isolated and characterized or subjected to further rounds of selection. Polymerases with altered substrate specificities will be kinetically and structurally characterized. A polymerase capable of the high fidelity incorporation of unnatural nucleobases into DNA would allow for the incorporation of increased diversity into nucleic acid libraries. A third base pair would also allow for the creation of additional codons, which would facilitate the incorporation of unnatural amino acids into proteins.
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