Oligonucleotides enable many biotechnological applications; however they are easily degraded by nucleases. Many modified nucleotides are degraded at decreased rates which improve oligonucleotide utility. Most applications of oligonucleotides rely on enzymatic synthesis. Unfortunately, native DNA polymerases do not recognize most useful modified nucleotide substrates. Directed evolution has been used to identify variants of Taq DNA polymerase I (Taq) that recognize substrates with 2' modifications. While mutant enzymes capable of modified nucleotide addition have been identified, to date, all of these enzymes are limited by their inability to synthesize full length modified DNA. Despite considerable efforts to evolve new activity there has been little work done to quantitatively characterize these evolved enzymes. Here, we describe efforts to comparatively and quantitatively characterize three enzymes previously evolved to recognize 2' modified substrates. Specifically, we have characterized the relationship between the number of modified nucleotides in the primer and the rate of modified nucleotide addition. This work identifies key enzymatic steps that limit these enzymes and has implications for the future design of Taq variants capable of synthesizing long 2' modified oligonucleotides.

Quantitative characterization of 2' modified primer extension by mutant Taq DNA polymerases

248th National Meeting (2014)

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