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.
Highlights
DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications
Enzymatic DNA synthesis strategies have been described that use protected nucleotide analogs and/or engineered polymerases to synthesize DNA with a precise sequence[18,19,20,21]
We describe a kinetically controlled system that uses terminal deoxynucleotidyl transferase (TdT) to catalyze the linkage of naturally occurring nucleoside triphosphates to synthesize customized DNA strands with short homopolymeric extensions
Summary
We determined the lowest required concentration for each nucleoside triphosphate that would result in extension of the initiator regardless of its terminal 3′ base The combination of these characterizations and optimizations yields a system where the addition of a series of nucleotides results in stepwise increases in the length of synthesized DNA (Fig. 1, Supplementary Fig. 7). The extension length for each added nucleoside triphosphate may vary, the resulting population of synthesized strands all share the same number and sequence of nucleotide transitions (Fig. 1b). These transitions between non-identical nucleotides encode user-defined information (Fig. 1c).
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