DNA is a valuable biotechnological tool; however, it is limited by its susceptibility to degradation by nucleases.  While 2’ modifications to DNA can prevent or slow nuclease degradation, most useful 2’ modified DNA modifications are not recognized by native DNA polymerases, limiting their application.  A number of researchers have used directed evolution to identify mutant enzymes of Taq DNA polymerase (Taq) capable of 2’ modified nucleoside triphosphates.  While several Taq mutants with improved recognition have been identified, these enzymes are unable to synthesize polymers of the 2’ modified DNA longer than 6-8 nucleotides.  Here, we describe our efforts to both characterize and engineer these previously evolved enzymes for improved properties.  We have characterized the ability of three previously evolved enzymes to recognize a range of 2’ modified nucleotides; through these studies, we have discovered new combinations of previously evolved enzymes-modified nucleotides as well as general, guiding principles for 2’ modified substrate recognition.  We have also biochemically characterized a number of mutants derived from the previously evolved mutants; from these studies, we have identified a minimal mutational motif (I614E/E615G) which imparts the highest unnatural function.  Finally, we present our preliminary efforts to apply our understanding to create mutant enzymes with improved recognition of 2’ modified nucleotides.

Characterization and development of Taq DNA polymerase mutants capable of synthesizing 2’ modified DNA

251st National Meeting (2016)

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