Photon-directed Multiplexed Enzymatic DNA Synthesis for Molecular Digital Data Storage

Abstract

New storage technologies are needed to keep up with the global demands of data generation. DNA is an ideal storage medium due to its stability, information density and ease of readout with advanced sequencing techniques. However, progress in writing DNA is stifled by the continued reliance on chemical synthesis methods. The enzymatic synthesis of DNA is a promising alternative, but thus far has not been well demonstrated in a highly parallelized manner. Here, we report a novel multiplexed enzymatic DNA synthesis method using maskless photolithography. Rapid uncaging of Co 2+ ions by patterned UV light activates Terminal deoxynucleotidyl Transferase (TdT) for spatially-selective synthesis on an array surface. Spontaneous quenching of reactions by the diffusion of excess caging molecules confines synthesisto light patterns and controls the extension length. We show that our multiplexed synthesis method can be used to store digital data by encoding 12 unique DNA oligonucleotide sequences with music from the 1985 Nintendo video game Super Mario Brothers TM , which is equivalent to 84 trits or 110 bits of data.In the era of Big Data, molecular DNA has become an increasingly attractive medium for the storage and archiving of digital data 1-6 . This is primarily attributed to its ultra-high storage density, which is currently estimated to be in the hundreds of petabytes per gram DNA 7 . The attractiveness of DNA as a data storage medium is additionally bolstered by its durability, longevity and energy efficiency compared to counterpart storage mediums; both analog and digital 8,9 . However, for storing a meaningful volume of digital data, the synthesis of many unique DNA sequences at long lengths is required. While advances in array-based Oligonucleotide Library Synthesis (OLS) technology have enabled highly multiplexed DNA oligonucleotide synthesis, production in this format still relies on decades old phosphoramidite chemical synthesis methods 10 . Many time-consuming steps of expensive and harsh reactions with the accumulation of toxic by-products greatly limit chemical synthesis as the demand for longer and larger quantities of DNA oligonucleotides increases 11 . 3 Recently, the use of terminal deoxynucleotidyl transferase (TdT), a template-independent polymerase, to synthesize DNA oligonucleotides was shown to be a promising alternative to chemical synthesis [12][13][14] . Because synthesis reactions are performed under aqueous conditions, many limiting aspects of the phosphoramidite chemistry can be circumvented or improved upon. This would be ideal for digital data storage in DNA; however, due to the natural promiscuity of TdT, controlling the enzyme in a sequencespecific manner is challenging [15][16][17] . In order to overcome this challenge, a recent study showed controlled TdT extension activity with apyrase by degrading free nucleotides needed for synthesis 14 .Incubation with optimized ratios of apyrase, TdT and nucleotide over multiple cycles resulted in the successful synthesis of several DNA oligonucleot...

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