Error-Prone DNA Synthesis on Click-Ligated Templates

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• Click ligation is a DNA fragment joining method using azide-alkyne cycloaddition.
• It forms a 4-methyl-1,2,3-triazole (trz) linkage, replacing phosphodiester bonds.
• The biological compatibility and DNA repair interactions of this trz linkage are largely unknown.

Purpose of the Study:

• To investigate the impact of the click ligation (trz) linkage on DNA repair pathways.
• To assess the behavior of various DNA polymerases when encountering the trz linkage.
• To determine the fidelity of DNA synthesis across the trz linkage.

Main Methods:

• Testing trz linkage resistance to human and bacterial endonucleases.
• Evaluating DNA polymerase stalling and bypass at the trz linkage using Pfu, DNA polymerase β, RB69 polymerase, and Klenow fragment.
• Assessing nucleotide misincorporation frequency across the trz linkage.

Main Results:

• The trz linkage demonstrated resistance to multiple DNA repair endonucleases.
• Significant polymerase stalling was observed with Pfu and DNA polymerase β.
• Phage RB69 polymerase and Klenow fragment showed bypass capabilities.
• High rates of dAMP misincorporation were detected for all polymerases except DNA polymerase β.

Conclusions:

• The trz linkage is resistant to common DNA repair mechanisms.
• Click ligation can cause significant DNA polymerase stalling and high-frequency miscoding.
• The potential for error generation limits the utility of click ligation in precise custom gene synthesis.