Probing and Enhancing DNA Ligase Specificity via Se‐Atom‐Mutagenesis of ATP for Accurate Gene Synthesis and Detection

Abstract Discriminating against base‐pair mismatches by nucleic acid‐related enzymes is essential for DNA replication, RNA replication, nucleic acid repair, transcription, and translation. However, the discrimination mechanisms at the atomic level remain poorly understood, limited by the availability of atom‐probing methods. Herein, we developed a single‐Se‐atom strategy (using ATPαSe) to explore mismatch discrimination in the DNA ligase system. We found that the Se‐atom (substituting O) in ATPαSe played a key role in the mismatch discrimination of DNA ligase, and ATPαSe significantly improved the ligation specificity, achieving up to 1000‐fold enhancement and expanding the region of mismatch discrimination up to 8 nt. Sequencing ligation products of various substrates generally confirmed the specificity enhancement using ATPαSe. Our mechanistic studies indicated that the specificity increase was correlated with the k cat reduction in mismatch ligations and with the disruption of the key interactions, supported by the site‐specific ligase mutations. Further, using ATPαSe, we achieved over 200‐fold increase in gene assembly efficiency and accomplished mismatch suppression in the single‐nucleotide‐polymorphism (SNP) detection. In conclusion, our Se‐atom‐probing strategy enables the atomic‐level studies on mismatch discrimination and offers a useful tool for accuracy enhancement, highlighting its broad potential for exploring molecular recognition and specificity.