Enzymatic Synthesis of Backbone-Modified Oligonucleotides Using T4 DNA Ligase

Abstract

T4 DNA ligase in high concentrations of certain crowding agents and cosolutes catalyzes the synthesis of a series of backbone-modified oligonucleotides that are difficult to obtain chemically. Backbone-modified nucleic acids are often enzymatically and chemically more stable, making them interesting as potential diagnostic or therapeutic agents, as a biosafety tool, or in nanotechnology. In this article, we describe a small-scale experiment to probe the efficiency of the ligation reaction of modified oligonucleotides in the presence of 3 M betaine and 10% PEG 8000, followed by large-scale ligation with subsequent isolation of the ligated oligonucleotide. The correct product formation can be verified using denaturing polyacrylamide gel electrophoresis and mass spectrometry. © 2019 by John Wiley & Sons, Inc.