Headpiece-assisted DNA data storage in solution and solid

Abstract

A headpiece was introduced in the construction of a DNA-based data storage platform. It was demonstrated that the involvement of the headpiece could largely improve the stability, recovery, resistance to DNA contamination, and accuracy in sequencing and data retrieval.

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We are living in an era of information explosion. It is predicted that the data production in the coming 2025 will be over 175 zettabytes.¹ It will be a great challenge to store the cosmic level information efficiently and safely using conventional storage media.^2,3 Due to multiple advantages including high density, longevity, energy efficiency, and sustainability, DNA data storage has been attracting more and more interest in academia and industry.^4,5 It is an upcoming consensus that DNA-based information storage will help to bridge the gap between data production and storage limitations, especially in archival storage or “cold information” storage.^6,7

DNA is a fragile biomolecule easy to degrade physically, chemically, or biologically⁸ from environmental intervention such as high temperature, ultraviolet light, irradiation, pH, reactive species, chemicals and solvents, biotic fluids, bacteria, and enzymes. Decades ago, palaeontologists and paleoanthropologists discovered that DNA can be stably stored in mineralized fossils for thousands of years, retaining the possibility for sequencing.⁹ It enlightened technologies to encapsulate DNA in an inorganic matrix to largely improve the stability and resistance to external damage.^10–12

Various materials have been applied for DNA encapsulation. For example, Grass and his collaborators¹³ have constructed DNA storage in glass (silica) particles, calcium phosphate, calcium chloride, magnesium chloride and so on,⁶ achieving high DNA loading, increased DNA stability, and simple sample handling.

The encapsulation strategy largely improved the stability of DNA in particles and salts. During the pre-treatments before encapsulation and ultimate PCR sequencing processes after DNA release, however, the stability of DNA will be a problem. Besides, chemicals and impurities are introduced during the encapsulation and releasing procedures. Further extraction and purification will be needed, involving additional losses of DNA. The release of DNA usually employs corrosive acids like hydrogen fluoride,¹⁴ which may damage the DNA molecules.

Moreover, we have noticed an essential problem which was rarely discussed by previous researchers, that is, external DNA contamination.^15,16 Most DNA storage approaches assumed their experiments were done in an ideal clean condition, without any potential microorganisms. However, the buffer applied in DNA experiments could be good for culturing bacteria and fungi,^17,18 introducing serious DNA contamination. Furthermore, inappropriate experimental operations would lead to human DNA contamination¹⁹ in data storage as well. DNA contaminations can extremely affect the accuracy of sequencing and information retrieval.

In this work, we attempted to address the several problems mentioned above in DNA data storage. Herein, a headpiece-assisted strategy was proposed to improve the stability, recovery, and resistance to DNA contamination in data storage (Fig. 1).

Fig. 1 Schematic illustration of storing and retrieving data in solution and solid phases in headpiece (HP)-assisted DNA data storage.

A headpiece (HP)²⁰ is a tri-branched polymer that covalently bonds the DNA duplex and provides a handle to a small-molecule ligand if required (Fig. S1 and S2, ESI†). HPs were widely used in DNA encoded libraries (DELs)^21,22 for new drug discoveries by pharmaceutical companies such as GSK.^23,24 They were verified to stabilize DNA duplexes and to be capable of ligand linkage in large chemical spaces. This work is the first attempt to involve them in DNA data storage, to the best of our knowledge.

Various experiments were constructed to test if the involvement of the headpiece could improve the stability, recovery, and resistance to DNA contamination in data storage as we expected. Considering the DNA duplex has two terminals in both directions, two different types of HP-DNA conjugate were designed (Fig. 2a and Fig. S3, ESI†), with one HP at one terminal (HP-Code), and two HPs at both terminals (HP-Code-HP), respectively. As a proof-of-concept test, we used a 57 bp DNA duplex (namely Code sequence) encoded with “Zheng Yi Ming Dao”, the motto of Shanghai Medical College, Fudan University, in ASCII codes.²⁵

Fig. 2 (a) The involvement of HPs improved DNA stability in solution and solid phases against environmental damage in data storage. Gel electrophoresis of different encoded DNAs (b) and RT-qPCR results (c) after being treated with 120 °C heating, enzymes, acid, base, ROS conditions, etc. before and after silica encapsulation.

First, gel electrophoresis and realtime fluorescence quantitative PCR (RT-qPCR) experiments²⁶ were applied to test the stabilization of HP-conjugated DNA sequences (Fig. 2b, c and Fig. S4, ESI†). Various environmental treatments including 120 °C heating for 30 min, enzymatic degradation including DNase I and exonuclease, acidic and basic treatments, and ROS production, were employed on nude Code DNA, HP-Code DNA, and HP-Code-HP DNA, respectively. For nude Code sequences without HP protection, strong degradation occurred after 120 °C heating, DNase I and acidic degradations. Weaker but significant degradation could be observed after exonuclease, ROS production and basic treatments. For HP-Code and HP-Code-HP DNAs, stabilizing effects of HP could be found in all environmental treatments, reflecting in the darker bands shown in gel electrophoresis images and higher qPCR yields. In all cases, HP-Code-HP showed better protection effects than HP-Code DNAs. We also demonstrated that the HP protection strategy was capable as a silica encapsulation strategy (Fig. 2c and Fig. S9, ESI†). There were no remarkable DNA losses for HP-Code and HP-Code-HP DNAs encapsulated in silica particles. Next generation sequencing results (Fig. S12 and S13, ESI†) supported that HP conjugation alone and with silica encapsulation would improve DNA stability. It can be concluded that HP could stabilize DNA in solution and solid phases.

As previously described, the tri-branched design of HP provided a handle for small-molecule ligand modification onto DNA duplexes.²⁷ That is, biotin can be easily attached to HP-Code and HP-Code-HP DNAs by facile amine condensation reactions (Fig. S10, ESI†). Thus, biotin–streptavidin affinity interactions^28,29 would be easily induced to improve the recovery of encoded DNA in etching-release processes for data storage in silica particles (Fig. 1). It is shown in Fig. S14 (ESI†) that the involvement of HPs is capable for biotinylating and further biotin–streptavidin affinity extraction. And the recovery of encoded DNA could be largely improved, especially for low DNA concentrations less than 10 ng μL⁻¹.

Further characterizations were employed to investigate the capability of HP conjugated DNA in solid phases via inorganic material encapsulation. We chose to follow Grass et al.'s classic silica encapsulation method with glass particles (Fig. 3a). After encapsulation, the silica particles were found to be around 80–200 nm in diameter from transmission electron microscopic (TEM) images and DLS (Fig. S5, ESI†). It was demonstrated from energy-dispersive X-ray spectroscopy (EDX) and scanning-transmission electron microscopy (STEM) high angle annular dark-field (HAADF) element mapping that the DNA strands were successfully encapsulated in silica particles (Fig. 3b and c). This was consistent with the results in ζ-potential, X-ray photoelectron spectra (XPS) and Fourier transform infrared spectrum (FT-IR) (Fig. S6–S8 and Table S1, ESI†). HP conjugated DNA could be undoubtedly encapsulated into silica particles, just like regular DNA strands.

Fig. 3 (a) Silica encapsulation and etching-release of encoded DNA. (b) Energy-dispersive X-ray spectra and (c) TEM and HAADF-STEM element mapping images of DNA-silica particles. Scale bar: 100 nm.

We have verified that HP conjugation can largely improve the stability and recovery for DNA data storage in solution and solid phases. Practical sequencing³⁰ and decoding procedures ought to be tested on HP conjugated DNA codes. HP-Code (Fig. 4a) and HP-Code-HP (Fig. 4b) DNAs in solution, and those released from silica particles using cold ethanol extraction and biotin–streptavidin affinity resin extraction, respectively, were employed for information decoding. It is shown in Fig. 4 that for all cases, including HP-Code and HP-Code-HP, for all conditions like in solution or extracted from etched silica particles using either cold ethanol extraction or biotin–streptavidin affinity extraction, the encoded data could be retrieved and decoded with high accuracy.

Fig. 4 (a) Sequencing results of HP-Code DNA strands in solution and those using cold ethanol extraction and streptavidin resin extraction after an encapsulation-release cycle. (b) Sequencing results of untreated HP-Code-HP DNA strands in solution, and those using cold ethanol extraction and streptavidin resin extraction after an encapsulation-release cycle. A: green, T: red, G: black, C: blue.

External DNA contamination experiments, where salmon sperm DNA (100 : 1) was applied as a mimetic of contaminated DNA, were carried out to estimate the resistance of the HP-conjugated DNA storage system. It could be seen that in the presence of external DNA contamination, the retrieving and decoding of the data were largely affected (Fig. 5a). It was impossible to directly retrieve the correct information from the sequencing result. Herein, the biotinylated HP-Code DNA showed powerful resistance to external DNA contamination. Biotin–streptavidin affinity extraction could be used to separate the encoded DNA from untagged contamination DNA strands. And the correct information was successfully retrieved from PCR sequencing at high accuracy (Fig. 5b).

Fig. 5 Sequencing results of encoded DNA in the presence of external DNA contamination (a) and biotin-HP conjugated DNA (b). A: green, T: red, G: black, C: blue. (c) Comparison of the properties of the four storage forms of DNA data storage systems with the conventional one in this work.

Finally, we summarized the advantages and disadvantages of the four different HP conjugating technologies, those are HP-Code and HP-Code-HP in solution, and HP-Code and HP-Code-HP in the solid state, respectively, and compared those to conventional nude Code DNA storage in solution (Fig. 5c). HP-Code and HP-Code-HP DNAs in solution exhibited improved stabilities, largely improved recoveries, but decreased PCR amplification efficiencies. The ultimate data retrieving accuracies showed improvements as well. The involvement of silica encapsulation greatly improved the stabilities of DNA in the solid state, but the recoveries of DNA and PCR amplification efficiencies declined as well. As to the ultimate data retrieving accuracies, it was demonstrated that silica encapsulation showed positive contributions in data decoding. It was observed that the HP-Code method was better than the HP-Code-HP one. It could be understood as conjugation of HPs at both terminals would hinder DNA unwinding and inhibit PCR amplification.

In summary, HP conjugation design was applied in DNA data storage in solution and solid phases. We verified that HP-conjugated DNA would improve the stability, recovery, resistance to external contamination, and data retrieval accuracy. It provided insights to develop DNA data storage capacitating covalent modification and inorganic encapsulation, expanding the toolbox for practical DNA storage. It will inspire polymer and inorganic chemists to construct future DNA information storage technology with improved performance.

The authors acknowledge support from the National Natural Science Foundation of China (22477017), the National Key R&D Program of China (2022YFC2804800) and the Science & Technology Commission of Shanghai Municipality (No. 23141900900).

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Notes and references

1. C. Q. Choi, ASEE Prism, 2020, 29, 22–25.
2. A. Doricchi, C. M. Platnich, A. Gimpel, F. Horn, M. Earle, G. Lanzavecchia, A. L. Cortajarena, L. M. Liz-Marzán, N. Liu, R. Heckel, R. N. Grass, R. Krahne, U. F. Keyser and D. Garoli, ACS Nano, 2022, 16, 17552–17571.
3. L. Ceze, J. Nivala and K. Strauss, Nat. Rev. Genet., 2019, 20, 456–466.
4. J. Koch, S. Gantenbein, K. Masania, W. J. Stark, Y. Erlich and R. N. Grass, Nat. Biotechnol., 2020, 38, 39–43.
5. C. Geng, S. Liu and X. Jiang, Chem. Sci., 2023, 14, 3973–3981.
6. A. X. Kohll, P. L. Antkowiak, W. D. Chen, B. H. Nguyen, W. J. Stark, L. Ceze, K. Strauss and R. N. Grass, Chem. Commun., 2020, 56, 3613–3616.
7. C. Bancroft, T. Bowler, B. Bloom and C. T. Clelland, Science, 2001, 293, 1763–1765.
8. C. Ottoni, B. Bekaert and R. Decorte, Taphonomy of Human Remains: Forensic Analysis of the Dead and the Depositional Environment, 2017, pp. 65–80.
9. L. Orlando, R. Allaby, P. Skoglund, C. Der Sarkissian, P. W. Stockhammer, M. C. Ávila-Arcos, Q. Fu, J. Krause, E. Willerslev, A. C. Stone and C. Warinner, Nat. Rev. Methods Prim., 2021, 1, 14.
10. P. L. Antkowiak, J. Koch, P. Rzepka, B. H. Nguyen, K. Strauss, W. J. Stark and R. N. Grass, Chem. Commun., 2022, 58, 3174–3177.
11. R. N. Grass, R. Heckel, M. Puddu, D. Paunescu and W. J. Stark, Angew. Chem., Int. Ed., 2015, 54, 2552–2555.
12. L. M. Wassermann, M. Scheckenbach, A. V. Baptist, V. Glembockyte and A. Heuer-Jungemann, Adv. Mater., 2023, 35, 2212024.
13. D. Paunescu, M. Puddu, J. O. B. Soellner, P. R. Stoessel and R. N. Grass, Nat. Protoc., 2013, 8, 2440–2448.
14. M. Puddu, D. Paunescu, W. J. Stark and R. N. Grass, ACS Nano, 2014, 8, 2677–2685.
15. A. Glassing, S. E. Dowd, S. Galandiuk, B. Davis and R. J. Chiodini, Gut Pathog., 2016, 8, 24.
16. B. Llamas, G. Valverde, L. Fehren-Schmitz, L. S. Weyrich, A. Cooper and W. Haak, STAR Sci. Technol. Archaeol. Res, 2017, 3, 1–14.
17. B. C. Millar, X. Jiru, J. E. Moore and J. A. P. Earle, J. Microbiol. Methods, 2000, 42, 139–147.
18. R. S. Tanner, Manual of Environmental Microbiology, 2007, pp. 69–78.
19. G. Jun, M. Flickinger, K. N. Hetrick, J. M. Romm, K. F. Doheny, G. R. Abecasis, M. Boehnke and H. M. Kang, Am. J. Hum. Genet., 2012, 91, 839–848.
20. G. Zhao, S. Zhong, G. Zhang, Y. Li and Y. Li, Angew. Chem., Int. Ed., 2022, 61, e202115157.
21. L. Jiang, S. Liu, X. Jia, Q. Gong, X. Wen, W. Lu, J. Yang, X. Wu, X. Wang, Y. Suo, Y. Li, M. Uesugi, Z. Qu, M. Tan, X. Lu and L. Zhou, J. Am. Chem. Soc., 2023, 145, 25283–25292.
22. A. A. Peterson and D. R. Liu, Nat. Rev. Drug Discovery, 2023, 22, 699–722.
23. P. A. Harris, B. W. King, D. Bandyopadhyay, S. B. Berger, N. Campobasso, C. A. Capriotti, J. A. Cox, L. Dare, X. Dong, J. N. Finger, L. C. Grady, S. J. Hoffman, J. U. Jeong, J. Kang, V. Kasparcova, A. S. Lakdawala, R. Lehr, D. E. McNulty, R. Nagilla, M. T. Ouellette, C. S. Pao, A. R. Rendina, M. C. Schaeffer, J. D. Summerfield, B. A. Swift, R. D. Totoritis, P. Ward, A. Zhang, D. Zhang, R. W. Marquis, J. Bertin and P. J. Gough, J. Med. Chem., 2016, 59, 2163–2178.
24. C. C. Arico-Muendel, MedChemComm, 2016, 7, 1898–1909.
25. K. T. Lua, Comput. Stand. Interfaces, 1990, 10, 117–124.
26. P. Y. Lee, J. Costumbrado, C. Y. Hsu and Y. H. Kim, J. Vis. Exp., 2012, e3923.
27. Y. Ding, G. J. Franklin, J. L. DeLorey, P. A. Centrella, S. Mataruse, M. A. Clark, S. R. Skinner and S. Belyanskaya, ACS Comb. Sci., 2016, 18, 625–629.
28. Z. Qu, Y. Zhang, Z. Dai, Y. Hao, Y. Zhang, J. Shen, F. Wang, Q. Li, C. Fan and X. Liu, Angew. Chem., Int. Ed., 2021, 60, 16693–16699.
29. L. J. Smith, R. C. Braylan, J. E. Nutkis, K. B. Edmundson, J. R. Downing and E. K. Wakeland, Anal. Biochem., 1987, 160, 135–138.
30. Z. Chen, W. Zhou, S. Qiao, L. Kang, H. Duan, X. S. Xie and Y. Huang, Nat. Biotechnol., 2017, 35, 1170–1178.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4cc05109b

‡ These authors contributed equally.

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