RecJ 5' Exonuclease Digestion of Oligonucleotide Failure Strands: A "Green" Method of Trityl-On Purification.

Abstract

Methods of error filtration and correction post-gene assembly are a major bottleneck in the synthetic biology pipeline. Current oligonucleotide purification strategies, including polyacrylamide gel electrophoresis and high-performance liquid chromatography, are often expensive and labor-intensive, give low mass recovery, and contain hazardous chemicals. To circumvent these limitations, we explored an enzymatic means of oligonucleotide purification using RecJ, which is the only known exonuclease to digest single-stranded DNA (ssDNA) in the 5' to 3' direction. As a potential application to remove failure strands generated in oligonucleotide synthesis, we found RecJ does not recognize the 5' dimethoxytrityl blocking group and could therefore be used to specifically target and digest unblocked failure strands. In combination with ssDNA binding protein (SSBP), which acts to recruit RecJ via C-terminal recognition, secondary structure formation is precluded, allowing for enhanced RecJ processivity. Using this method to purify crude trityl-on oligonucleotides, we also found on average 30 units of RecJ with 0.5 μg of SSBP digests 53 pmol of 5' hydroxylated ssDNA (60 min at 37 °C). With these parameters, the average purity is increased by 8%. As such, this novel method can be adapted to most laboratory practices, particularly those with DNA synthesis automation as a simple, inexpensive (<$4), and eco-friendly means of oligonucleotide trityl-on purification.