A DNA Polymerase with Increased Reactivity for Ribonucleotides and C5-Modified Deoxyribonucleotides

Abstract

DNA polymerases find broad applications in molecular biology techniques (e.g., polymerase chain reaction, genome sequencing, or diagnostic methods).[1] Recently, DNA polymerases have increasingly been employed to synthesize highly functionalized DNA and thereby show great promise for future applications ranging from new DNA sequencing technologies[2] to diagnostics,[3] SELEX,[4] and DNA-based nanotechnology.[5] Moreover, it was shown that nucleotide analogues with a simplified sugar backbone such as threosyl or glyceryl surrogates are polymerized by DNA polymerases.[6] Interestingly, these reports show that DNA polymerases from sequence family B are superior to other DNA polymerases in polymerizing highly modified nucleotides. The A485L mutant of Thermococcus species 98N DNA polymerase termed Therminator DNA polymerase (Therminator pol)[7] was widely applied due to its ability to accept several unnatural nucleoside triphosphate analogues as substrates to some extent. However, the observed low efficiency of the enzyme in the processing of several modified substrates often still prevents further applications. Directed evolution shows great promise for improving the performance of DNA polymerases.[8] Here we present the generation of a new Therminator pol variant with broadened substrate scope. The enzyme was identified by a screen of a Therminator pol library that was generated by error-prone PCR. We show that the variant is able to polymerize sugar as well as nucleobase-modified nucleotides to a greater extent than the parental enzyme.