DNA is increasingly being explored as an alternative medium for digital information storage, but the potential information loss from degradation and associated issues with error during reading challenge its wide-scale implementation. To address this, we propose an atomic-scale encoding standard for DNA, where information is encoded in degradation-resistant analogues of natural nucleic acids (xNAs). To better enable this approach, we used directed evolution to create a polymerase capable of transforming 2'-O-methyl templates into double-stranded DNA. Starting from a thermophilic, error-correcting reverse transcriptase, RTX, we evolved an enzyme (RTX-Ome v6) that relies on a fully functional proofreading domain to correct mismatches on DNA, RNA, and 2'-O-methyl templates. In addition, we implemented a downstream analysis strategy that accommodates deletions that arise during phosphoramidite synthesis, the most common type of synthesis error. By coupling and integrating new chemistries, enzymes, and algorithms, we further enable the large-scale use of nucleic acids for information storage.
Keywords: 2′-O-methyl; DNA data storage; directed evolution; nucleic acid analogues; protein engineering; reverse transcriptase.