Terminator-free template-independent enzymatic DNA synthesis for digital information storage

TLDR

DNA is an emerging medium for digital data, and its adoption can be accelerated by synthesis processes specialized for storage. The study presents a de novo enzymatic synthesis strategy for data storage using template‑independent TdT under kinetically controlled conditions. The method encodes data in nucleotide transitions, generating short homopolymeric extensions by iterative substrate addition with lengths regulated by apyrase‑mediated degradation. The authors synthesized 144‑bit DNA strands, retrieved them via streaming nanopore sequencing, and showed that a digital codec enables robust data recovery from imperfect synthesis, demonstrating the potential of distributive enzymatic synthesis for DNA storage.

Abstract

Abstract DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA.