Synthesis of threose nucleic acid (TNA) triphosphates and oligonucleotides by polymerase-mediated primer extension.

Abstract

This unit describes the chemical synthesis of α-L-threofuranosyl nucleic acid (TNA) triphosphates for thymidine (T), guanosine (G), cytidine (C), and the diaminopurine (D) analog of adenosine and their incorporation into TNA oligonucleotides by enzyme-mediated polymerization of a DNA primer-template complex. Starting from suitably protected threofuranosyl nucleosides, TNA triphosphates are synthesized in a single-pot reaction and purified by ion-exchange and HPLC chromatography. Purified TNA triphosphates are diluted into stock solutions and used as substrates for the synthesis of TNA oligonucleotides. Oligonucleotide synthesis is accomplished using Therminator DNA polymerase, a commercial variant of the 9(o)N DNA polymerase bearing the A485L mutation.