Enzymatic Synthesis of Phosphonomethyl Oligonucleotides by Therminator Polymerase

Abstract

Projects in synthetic biology imply the study of the substrate specificity of natural and non-natural biopolymers with enzymatic activity and the identification of the products that are obtained. Herein we describe the enzymatic synthesis of 3’–2’ phosphonomethyl–threosyl and 5’–3’ phosphonomethyl–deoxyribosyl oligonucleotides by Therminator polymerase (Scheme 1). We demonstrate that phosphonate nucleotides can be polymerized by this enzyme with the formation of oligonucleotides with more than sixinternucleotide bonds. The enzymes that catalyze the formation of the 5’–3’ phosphodiester bond in DNA and RNA are polymerases and use nucleoside triphosphates as substrates. Therminator polymerase is a mutant variant of the 98 Ne x o polymerase (Thermococcus species 98N-7), in which the Ala 485 residue has been replaced with a Leu residue. With this mutation in an a helixthat is oriented away from the nucleotide-binding site, Therminator polymerase possesses an enhanced ability to incorporate modified substrates, such as dideoxynucleosides, ribonucleosides, and acyclic nucleosides, by using their triphosphate moiety as a substrate. [1–3] We envisaged the synthesis of 3’–2’ phosphonomethyl–