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Cited by 121DNA and Biological ComputingAdvanced biosensing and bioanalysis techniquesAlgorithms and Data Compression

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

Open access
Tabatabaei, S. Kasra, Wang, Boya, Athreya, Nagendra Bala Murali, Enghiad, Behnam, Hernandez, Alvaro Gonzalo, Fields, Christopher J., Leburton, Jean-Pierre, Soloveichik, David, Zhao, Huimin, Milenkovic, Olgica·2020·Nature Communications
DOI 10.1038/s41467-020-15588-zPMC PMC7142088
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Abstract

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.

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Crossref · 10.1038/s41467-020-15588-z

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Figures

Fig. 1 The native DNA-based data storage platform. In the Write component, arbitrary user content is converted into a binary message. The message is then parsed into blocks of m bits, where m corresponds to the number of nicking positions on the register (for the running example, m = 10). Different (orthogonal) registers may be used to encode information in parallel, as indicated by the different colors of the DNA strings. Subsequently, binary information is translated into positional information indicating where to nick. Nicking reactions are performed in parallel via combinations of PfAgo and guides. In the Read component, nicked products are purified and denatured to obtain a pool of ssDNAs of different lengths. The pool of ssDNAs is sequenced via MiSeq either as part of an orthogonal register mixture or individually. The output reads are processed by first performing reference-based alignment of the reads, and then using read coverages to determine the nicked positions.
Fig. 1 The native DNA-based data storage platform. In the Write component, arbitrary user content is converted into a binary message. The message is then parsed into blocks of m bits, where m corresponds to the number of nicking positions on the register (for the running example, m = 10). Different (orthogonal) registers may be used to encode information in parallel, as indicated by the different colors of the DNA strings. Subsequently, binary information is translated into positional information indicating where to nick. Nicking reactions are performed in parallel via combinations of PfAgo and guides. In the Read component, nicked products are purified and denatured to obtain a pool of ssDNAs of different lengths. The pool of ssDNAs is sequenced via MiSeq either as part of an orthogonal register mixture or individually. The output reads are processed by first performing reference-based alignment of the reads, and then using read coverages to determine the nicked positions.CC BY · PubMed Central
Fig. 2 Writing and reading the encoded data. a PfAgo can nick several pre-designated locations on only one strand (left, top) or both strands (left, bottom), simultaneously. In the first register, the stored content is 110…1, while in the second register, the content is 121…0. The chosen register is a PCR product of a 450 bp E. coli genomic DNA fragment with 10 pre-designated non-uniformly spaced nicking positions. The positional code 238 corresponds to the binary vector 0110000100 (right). b The MiSeq sequencing reads were aligned to the reference register to determine the positions of the nicks. The size distribution histogram (right) and coverage plots (left) are then generated based on the frequency and coverage depth of the reads. Coverage plots allow for straightforward detection of nicked and unnicked sites. In the example shown, all the ten positions were nicked, resulting in eleven aligned fragments. Source data are available in the Source Data file.
Fig. 2 Writing and reading the encoded data. a PfAgo can nick several pre-designated locations on only one strand (left, top) or both strands (left, bottom), simultaneously. In the first register, the stored content is 110…1, while in the second register, the content is 121…0. The chosen register is a PCR product of a 450 bp E. coli genomic DNA fragment with 10 pre-designated non-uniformly spaced nicking positions. The positional code 238 corresponds to the binary vector 0110000100 (right). b The MiSeq sequencing reads were aligned to the reference register to determine the positions of the nicks. The size distribution histogram (right) and coverage plots (left) are then generated based on the frequency and coverage depth of the reads. Coverage plots allow for straightforward detection of nicked and unnicked sites. In the example shown, all the ten positions were nicked, resulting in eleven aligned fragments. Source data are available in the Source Data file.CC BY · PubMed Central
Fig. 3 Multi-register and sense-antisense recording. a Five orthogonal registers used instead of one single register. Each vertical section represents one register in genome dictated reading order, and each row shows the read lengths retrieved after sequencing analysis. Read lengths are recorded on the left and sequencing depths on the right axis. b Nicking both the sense and antisense strands. Here, the binary format is switched to ternary format, i.e., for each nicking site, a nick on the sense strand denotes 1, a nick on the antisense denotes 2, and no nick denotes 0. The coverage (left) and size distribution (right) plots verify that the register can be nicked on ten prescribed sites on both strands, six of which are located on the sense strand and four on the antisense strands, corresponding to the 10-bit string of data: 1212121211. The sense-antisense nicking approach may also be used for parallel recording on multiple registers. Source data are available in the Source Data file.
Fig. 3 Multi-register and sense-antisense recording. a Five orthogonal registers used instead of one single register. Each vertical section represents one register in genome dictated reading order, and each row shows the read lengths retrieved after sequencing analysis. Read lengths are recorded on the left and sequencing depths on the right axis. b Nicking both the sense and antisense strands. Here, the binary format is switched to ternary format, i.e., for each nicking site, a nick on the sense strand denotes 1, a nick on the antisense denotes 2, and no nick denotes 0. The coverage (left) and size distribution (right) plots verify that the register can be nicked on ten prescribed sites on both strands, six of which are located on the sense strand and four on the antisense strands, corresponding to the 10-bit string of data: 1212121211. The sense-antisense nicking approach may also be used for parallel recording on multiple registers. Source data are available in the Source Data file.CC BY · PubMed Central
Fig. 4 Non-destructive bitwise random access. a Non-destructive detection of toeholds through a fluorophore and quencher labeled Reporter strand. Once the Reporter hybridizes with the toehold on the register strand, a fluorescence signal is observed due to the increase of the distance between the fluorophore and quencher. The Reporter strand can be pulled off from the register once the Reporter* strand hybridizes with the Reporter. b Kinetics of detecting the concentrations of registers with and without toeholds in a mixtures (left). The fluorescence signals saturate within 20 min. The samples were mixed no more than 2 min before measurement. The concentration of toehold-ed DNA can be accurately quantified through fluorescence intensity (right), as it increases linearly with the concentration of the registers with toehold. c PAGE gel results for non-destructive detection of a toehold. Left: the gel was not stained with other fluorescence dyes and hence only the species with self-fluorescence are observed. After adding the Reporter, a large size complex appears in the lane “Template+Reporter”, indicating hybridization of the Reporter and the register. After the Reporter* is added, as seen in the lane “Template+Reporter+Reporter*”, the large size complex in the previous lane no longer exhibits self-fluorescence, indicating that the Reporter strand is pulled off from the register. Right: The same gel after staining (with SYBR gold: ThermoFisher Scientific). Note that the same molecular components are observed, in addition to the Reporter*. The PAGE analysis was performed once as this is a standard molecular computing setup and the results are expected to be consistent over many trials. Source data is available in the Source Data file.
Fig. 4 Non-destructive bitwise random access. a Non-destructive detection of toeholds through a fluorophore and quencher labeled Reporter strand. Once the Reporter hybridizes with the toehold on the register strand, a fluorescence signal is observed due to the increase of the distance between the fluorophore and quencher. The Reporter strand can be pulled off from the register once the Reporter* strand hybridizes with the Reporter. b Kinetics of detecting the concentrations of registers with and without toeholds in a mixtures (left). The fluorescence signals saturate within 20 min. The samples were mixed no more than 2 min before measurement. The concentration of toehold-ed DNA can be accurately quantified through fluorescence intensity (right), as it increases linearly with the concentration of the registers with toehold. c PAGE gel results for non-destructive detection of a toehold. Left: the gel was not stained with other fluorescence dyes and hence only the species with self-fluorescence are observed. After adding the Reporter, a large size complex appears in the lane “Template+Reporter”, indicating hybridization of the Reporter and the register. After the Reporter* is added, as seen in the lane “Template+Reporter+Reporter*”, the large size complex in the previous lane no longer exhibits self-fluorescence, indicating that the Reporter strand is pulled off from the register. Right: The same gel after staining (with SYBR gold: ThermoFisher Scientific). Note that the same molecular components are observed, in addition to the Reporter*. The PAGE analysis was performed once as this is a standard molecular computing setup and the results are expected to be consistent over many trials. Source data is available in the Source Data file.CC BY · PubMed Central

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Research works printed but not linked: 2

  1. Wang, B., Chalk, C. & Soloveichik, D. in DNA Computing and Molecular Programming. DNA 2019. Lecture Notes in Computer Science, Vol. 11648 (eds. Thachuk, C. & Liu, Y.) (Springer, Cham, 2019).
  2. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. ArXiv e-prints, 1303 (2013).

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Metadata

Authors
Tabatabaei, S. K., Wang, B., Athreya, N. B. M., Enghiad, B., Hernandez, A. G., Fields, C. J., Leburton, J.-P., Soloveichik, D., Zhao, H., & Milenkovic, O.
Affiliations
  • University of Illinois Urbana-Champaign (US)
  • The University of Texas at Austin (US)
Corresponding author
Zhao, Huimin
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-04-08
Volume
11
Issue
1
Pages
1742-1742
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-020-15588-z
PMC
PMC7142088
OpenAlex
W3015846461
Cited by
120
References
41
Citations by year
2026: 13 · 2025: 15 · 2024: 12 · 2023: 25 · 2022: 15 · 2021: 25 · 2020: 14
FWCI
6.41
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
United States Department of Defense | Defense Advanced Research Projects Agency (W911NF-18-2-0032)
Topics
  • Physical Sciences › Mathematics › Theoretical Computer Science › DNA and Biological Computing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
  • Physical Sciences › Mathematics › Theoretical Computer Science › Algorithms and Data Compression
Keywords
DNA data storage, DNA nicking, Argonaute proteins, Pyrococcus furiosus, in-memory computing, DNA topology
MeSH terms
Base Sequence, DNA, Sequence Analysis, DNA, Pyrococcus furiosus, Argonaute Proteins

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