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

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.