Evolved DNAzymes and Stable Activation Chemistry Enable High‐Efficiency DNA Ligation

ABSTRACT DNA ligation is a fundamental reaction used in a variety of applications, typically performed by T4 DNA ligase. For certain applications, such as DNA data storage, DNA‐ligating DNAzymes offer a more stable and cost‐effective alternative to protein enzymes. The E47 DNAzyme is among the most efficient DNA‐ligating DNAzymes reported, but it still lacks sufficient activity for widespread adoption and requires a highly unstable phosphoimidazole‐activated DNA substrate to function. In this study, we performed in vitro selection using a pre‐structured library based on E47 and identified sequences with more than a twofold increase in ligation activity, representing the fastest DNA‐ligating DNAzymes reported to date. We also screened alternative imidazolide compounds for substrate activation and found that phosphobenzimidazole‐activated DNA substrates are significantly more stable, remaining intact for at least 24 h at room temperature. These advances improve the practicality of DNAzymes for ligation‐based applications and broaden their potential use in DNA data storage.