Limitations of modified substrate recognition by Taq DNA polymerase mutants (967.2)

Abstract

DNA Polymerase, which is required for enzymatic DNA synthesis, does not recognize or incorporate 2’ modified substrates although they are useful in a number of biotechnology applications. Through directed evolution, mutant polymerases have been identified with the ability to incorporate 2’ modified substrates; however, they are incapable of full primer extension limiting their use. Here we compare the ability of 5 previously evolved enzymes to extend with 2’OH and 2’OMe modified nucleotides. Comparative studies reveal stark differences in activity amongst previously evolved mutants. In particular, we observe that while all of the enzymes are capable of incorporating a modified nucleotide onto an unmodified primer with moderate to high efficiency, there are significant differences between enzymes in the rate of incorporation of additional modified nucleotides onto the modified primers. Understanding the limitations of modified substrate incorporation, in conjunction with other mutant polymerase characterization currently being done, will help guide future endeavors to rationally design mutant polymerases that can overcome these extension limitations.