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Bioorthogonal information storage in l-DNA with a high-fidelity mirror-image Pfu DNA polymerase

Abstract

Natural DNA is exquisitely evolved to store genetic information. The chirally inverted l-DNA, possessing the same informational capacity but resistant to biodegradation, may serve as a robust, bioorthogonal information repository. Here we chemically synthesize a 90-kDa high-fidelity mirror-image Pfu DNA polymerase that enables accurate assembly of a kilobase-sized mirror-image gene. We use the polymerase to encode in l-DNA an 1860 paragraph by Louis Pasteur that first proposed a mirror-image world of biology. We realize chiral steganography by embedding a chimeric d-DNA/l-DNA key molecule in a d-DNA storage library, which conveys a false or secret message depending on the chirality of reading. Furthermore, we show that a trace amount of an l-DNA barcode preserved in water from a local pond remains amplifiable and sequenceable for 1 year, whereas a d-DNA barcode under the same conditions could not be amplified after 1 day. These next-generation mirror-image molecular tools may transform the development of advanced mirror-image biology systems and pave the way for the realization of the mirror-image central dogma and exploration of their applications.

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Fig. 1: Synthetic natural and mirror-image Pfu DNA polymerases.
Fig. 2: Mirror-image gene assembly by the mirror-image Pfu DNA polymerase.
Fig. 3: Mirror-image DNA information storage.
Fig. 4: Chiral steganography.
Fig. 5: Mirror-image DNA barcoding of environmental water samples.

Data availability

The data that support the findings of this study are available within the paper and its Supplementary Information. Metagenomic sequencing data are available at the National Center for Biotechnology Information under the BioProject number PRJNA707266.

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Acknowledgements

We thank J. Chen, M. Chen, W. Jiang, J. J. Ling, G. Wang, Y. Xu and R. Zhao for assistance with the experiments, and W. Jiang, M. J. McFall-Ngai, Y. Shi, J. W. Szostak, H. W. Wang, E. Winfree and N. Yan for comments on the manuscript. T.F.Z. was supported by funding from the National Natural Science Foundation of China (21925702, 32050178 and 21750005), the Tsinghua-Peking Center for Life Sciences, the Tencent Foundation, the Beijing Advanced Innovation Center for Structural Biology and the Beijing Frontier Research Center for Biological Structure.

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Contributions

C.F. performed the chemical synthesis. Q.D. performed the biochemistry experiments. All authors analyzed and discussed the results. T.F.Z. designed and supervised the study, and wrote the paper.

Corresponding author

Correspondence to Ting F. Zhu.

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Peer review information Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Design of the mutant Pfu-N fragment.

a, Mutant Pfu-N fragment amino acid sequence with an N-terminal His6 tag and 4 point mutations (E102A, E276A, K317G, V367L, in parentheses) to introduce additional NCL sites. In addition, 25 isoleucine residues (underlined) were substituted to facilitate the chemical synthesis and reduce the synthesis costs for the mirror-image version. Colors of the amino acid sequences correspond to the peptide segment colors used in panel b. b, Synthetic route for synthesizing the mutant Pfu-N fragment.

Extended Data Fig. 2 Design of the mutant Pfu-C fragment.

a, Mutant Pfu-C fragment amino acid sequence with 1 point mutation (I540A, in parentheses) to introduce an additional NCL site. In addition, 16 isoleucine residues (underlined) were substituted to facilitate the chemical synthesis and reduce the synthesis costs for the mirror-image version. Colors of the amino acid sequences correspond to the peptide segment colors used in panel b. b, Synthetic route for synthesizing the mutant Pfu-C fragment.

Extended Data Fig. 3 Mirror-image DNA information storage.

a, Design of information-storing L-DNA segments. Caret, uppercase. b, Experimental procedures for mirror-image DNA information storage.

Supplementary information

Supplementary Information

Supplementary Figs. 1–60 and Tables 1–7.

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Fan, C., Deng, Q. & Zhu, T.F. Bioorthogonal information storage in l-DNA with a high-fidelity mirror-image Pfu DNA polymerase. Nat Biotechnol 39, 1548–1555 (2021). https://doi.org/10.1038/s41587-021-00969-6

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