Chemical modifications to DNA, such as 2' modifications, are expected to increase the biotechnological utility of DNA; however, these modified forms of DNA are limited by their inability to be effectively synthesized by DNA polymerase enzymes. Previous efforts have identified mutant Thermus aquaticus DNA polymerase I (Taq) enzymes capable of recognizing 2'-modified DNA nucleotides. While these mutant enzymes recognize these modified nucleotides, they are not capable of synthesizing full length modified DNA; thus, further engineering is required for these enzymes. Here, we describe comparative biochemical studies that identify useful, but previously uncharacterized, properties of these enzymes; one enzyme, SFM19, is able to recognize a range of 2'-modified nucleotides much wider than that previously examined, including fluoro, azido, and amino modifications. To understand the molecular origins of these differences, we also identify specific amino acids and combinations of amino acids that contribute most to the previously evolved unnatural activity. Our data suggest that a negatively charged amino acid at 614 and mutation of the steric gate residue, E615, to glycine make up the optimal combination for modified oligonucleotide synthesis. These studies yield an improved understanding of the mutational origins of 2'-modified substrate recognition as well as identify SFM19 as the best candidate for further engineering, whether via rational design or directed evolution.
DNA polymerases enable key DNA biotechnologies by performing enzymatic amplification of DNA with high efficiency and fidelity, enabling DNA's use in polymerase chain reaction (PCR), sequencing techniques and many different applications in therapeutics and diagnostics. While these astounding properties of DNA polymerases enable the use of these technologies, they also restrict them because they do not recognize modified substrates. Three previous studies have evolved DNA pol I from Thermus aquaticus (Taq) to recognize 2’ modifications of DNA; while these have resulted in an increase in modified substrate recognition, these enzymes are not active enough for practical use. Here, we describe efforts to identify the key mutations that impart unnatural activity on these previously identified enzymes. In particular, we have focused on mutations at I614 and E615 since they occur in all three previously identified mutants. Data show that much of the activity that is present in the previously evolved enzymes is largely a result of the E615G mutation. Mutations at I614 have been found to have no effect when present alone, but have also been found to have varying effects on enzymatic activity when in conjunction with the E615 mutation. Current work is also being done to further investigate the synergistic interactions of mutations of I614 and E615G. These efforts will elucidate key features of the evolution of unnatural substrate recognition by mutant Taq enzymes and will aid in future polymerase engineering efforts.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.