Mutant Taq polymerase recognition of 2’ modified nucleotides (967.1)

Abstract

Mutant Taq DNA Polymerases with the ability to recognize modified nucleotides at the 2’ sugar position have been isolated through directed evolution; these nucleotide modifications increase DNA’s utility by preventing degradation in biological systems. However, these previously identified mutants are only capable of extending DNA templates by two to six base pairs with modified nucleotides. This project focuses on characterizing the increased promiscuity of these enzymes and their ability to recognize a greater substrate range beyond the nucleotides used for their selection. Three previously evolved mutants are able to recognize a surprisingly wide range of 2’ modified nucleotides (dNTP, O‐Me, F, NH3, N3, OH) with varying efficiencies, providing insight into possible new enzyme‐substrate pairings. We describe initial kinetic characterization and further engineering efforts, which may lead to valuable new mutant enzyme‐modified substrate pairs capable of full length synthesis.