Molecular data storage is an attractive alternative for dense and durable information storage, which is sorely needed to deal with the growing gap between information production and the ability to store data. DNA is a clear example of effective archival data storage in molecular form. In this Review, we provide an overview of the process, the state of the art in this area and challenges for mainstream adoption. We also survey the field of in vivo molecular memory systems that record and store information within the DNA of living cells, which, together with in vitro DNA data storage, lie at the growing intersection of computer systems and biotechnology.

Molecular digital data storage using DNA

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. Adoption of DNA as a data storage medium could be accelerated with specialized synthesis processes and codecs. The authors describe TdT-mediated DNA synthesis in which data is stored in transitions between non-identical nucleotides and the use of synchronization markers to provide error tolerance.

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

The density and long-term stability of DNA make it an appealing storage medium, particularly for long-term data archiving. Existing DNA storage technologies involve the synthesis and sequencing of multiple nominally identical molecules in parallel, resulting in information redundancy. We report the development of encoding and decoding methods that exploit this redundancy using composite DNA letters. A composite DNA letter is a representation of a position in a sequence that consists of a mixture of all four DNA nucleotides in a predetermined ratio. Our methods encode data using fewer synthesis cycles. We encode 6.4 MB into composite DNA, with distinguishable composition medians, using 20% fewer synthesis cycles per unit of data, as compared to previous reports. We also simulate encoding with larger composite alphabets, with distinguishable composition deciles, to show that 75% fewer synthesis cycles are potentially sufficient. We describe applicable error-correcting codes and inference methods, and investigate error patterns in the context of composite DNA letters. Toward more storage for less synthesis using a six-letter composite DNA alphabet.

Data storage in DNA with fewer synthesis cycles using composite DNA letters

Measuring biological data across time and space is critical for understanding complex biological processes and for various biosurveillance applications However, such data are often inaccessible or difficult to directly obtain Less invasive, more robust and higher-throughput biological recording tools are needed to profile cells and their environments DNA-based cellular recording is an emerging and powerful framework for tracking intracellular and extracellular biological events over time across living cells and populations Here, we review and assess DNA recorders that utilize CRISPR nucleases, integrases and base-editing strategies, as well as recombinase and polymerase-based methods Quantitative characterization, modelling and evaluation of these DNA-recording modalities can guide their design and implementation for specific application areas

DNA-based memory devices for recording cellular events.

Synthetic DNA-based data storage systems have received significant attention due to the promise of ultrahigh storage density and long-term stability. However, all known platforms suffer from high cost, read-write latency and error-rates that render them noncompetitive with modern storage devices. One means to avoid the above problems is using readily available native DNA. As the sequence content of native DNA is fixed, one can modify the topology instead to encode information. Here, we introduce DNA punch cards, a macromolecular storage mechanism in which data is written in the form of nicks at predetermined positions on the backbone of native double-stranded DNA. The platform accommodates parallel nicking on orthogonal DNA fragments and enzymatic toehold creation that enables single-bit random-access and in-memory computations. We use Pyrococcus furiosus Argonaute to punch files into the PCR products of Escherichia coli genomic DNA and accurately reconstruct the encoded data through high-throughput sequencing and read alignment. Current synthetic DNA-based data storage systems have high recording costs, read-write latency and error-rates that make them uncompetitive compared to traditional digital storage. The authors use nicks in native DNA to encode data in parallel and create access sites for in-memory computations.

DNA punch cards for storing data on native DNA sequences via enzymatic nicking

Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

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De novo DNA synthesis using polymerase-nucleotide conjugates

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.

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Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis.

Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Nucleic acid synthesis and sequencing using tethered nucleoside triphosphates

Novel enzymatic methods are poised to become the dominant processes for de novo synthesis of DNA, promising functional, economic, and environmental advantages over the longstanding approach of phosphoramidite synthesis. Before this can occur, however, enzymatic synthesis methods must be parallelized to enable production of multiple DNA sequences simultaneously. As a means to this parallelization, we report a polymerase-nucleotide conjugate that is cleaved using electrochemical oxidation on a microelectrode array. The developed conjugate maintains polymerase activity toward surface-bound substrates with single-base control and detaches from the surface at mild oxidative voltages, leaving an extendable oligonucleotide behind. Our approach readies the way for enzymatic DNA synthesis on the scale necessary for DNA-intensive applications such as DNA data storage or gene synthesis.

https://pubs.acs.org/doi/pdf/10.1021/acssynbio.3c00044

Sebastian Palluk

Papers

De novo DNA synthesis using polymerase-nucleotide conjugates

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis.

Nucleic acid synthesis and sequencing using tethered nucleoside triphosphates

Spatially Selective Electrochemical Cleavage of a Polymerase-Nucleotide Conjugate