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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Biotechnology Open Access 06 June 2022
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Pasteur, L. Researches on the Molecular Asymmetry of Natural Organic Products (Société Chimique de Paris, 1860) Reprint No. 14 (Alembic Club, 1905).
Wang, Z., Xu, W., Liu, L. & Zhu, T. F. A synthetic molecular system capable of mirror-image genetic replication and transcription. Nat. Chem. 8, 698–704 (2016).
Peplow, M. Mirror-image enzyme copies looking-glass DNA. Nature 533, 303–304 (2016).
Peplow, M. A conversation with Ting Zhu. ACS Cent. Sci. 4, 783–784 (2018).
Beaucage, S. L. & Caruthers, M. H. Deoxynucleoside phosphoramidites - a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22, 1859–1862 (1981).
Liu, Y. et al. Synthesis and applications of RNAs with position-selective labelling and mosaic composition. Nature 522, 368–372 (2015).
Merrifield, R. B. Solid phase peptide synthesis. 1. Synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149–2154 (1963).
Dawson, P., Muir, T., Clark-Lewis, I. & Kent, S. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994).
Yan, L. Z. & Dawson, P. E. Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J. Am. Chem. Soc. 123, 526–533 (2001).
Fang, G.-M. et al. Protein chemical synthesis by ligation of peptide hydrazides. Angew. Chem. Int. Ed. Engl. 50, 7645–7649 (2011).
Milton, R., Milton, S. & Kent, S. Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity. Science 256, 1445–1448 (1992).
Zawadzke, L. E. & Berg, J. M. A racemic protein. J. Am. Chem. Soc. 114, 4002–4003 (1992).
Weinstock, M. T., Jacobsen, M. T. & Kay, M. S. Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity. Proc. Natl Acad. Sci. USA 111, 11679–11684 (2014).
Vinogradov, A. A., Evans, E. D. & Pentelute, B. L. Total synthesis and biochemical characterization of mirror image barnase. Chem. Sci. 6, 2997–3002 (2015).
Xu, W. et al. Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction. Cell Discov. 3, 17008 (2017).
Jiang, W. et al. Mirror-image polymerase chain reaction. Cell Discov. 3, 17037 (2017).
Pech, A. et al. A thermostable d-polymerase for mirror-image PCR. Nucleic Acids Res. 45, 3997–4005 (2017).
Hartrampf, N. et al. Synthesis of proteins by automated flow chemistry. Science 368, 980–987 (2020).
Wang, M. et al. Mirror-image gene transcription and reverse transcription. Chem 5, 848–857 (2019).
Lamarche, B. J., Kumar, S. & Tsai, M. D. ASFV DNA polymerase X is extremely error-prone under diverse assay conditions and within multiple DNA sequence contexts. Biochemistry 45, 14826–14833 (2006).
Ling, H., Boudsocq, F., Woodgate, R. & Yang, W. Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication. Cell 107, 91–102 (2001).
Boudsocq, F., Iwai, S., Hanaoka, F. & Woodgate, R. Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic polη. Nucleic Acids Res. 29, 4607–4616 (2001).
Cline, J., Braman, J. C. & Hogrefe, H. H. PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24, 3546–3551 (1996).
Tang, S. et al. Practical chemical synthesis of atypical ubiquitin chains by using an isopeptide-linked Ub isomer. Angew. Chem. Int. Ed. Engl. 56, 13333–13337 (2017).
Sun, H. & Brik, A. The journey for the total chemical synthesis of a 53 kDa protein. Acc. Chem. Res. 52, 3361–3371 (2019).
Hansen, C. J., Wu, L., Fox, J. D., Arezi, B. & Hogrefe, H. H. Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide ɣ-phosphate derivative. Nucleic Acids Res. 39, 1801–1810 (2011).
Wan, Q. & Danishefsky, S. J. Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew. Chem. Int. Ed. Engl. 46, 9248–9252 (2007).
Hyde, C., Johnson, T., Owen, D., Quibell, M. & Sheppard, R. Some ‘difficult sequences’ made easy. Int. J. Pept. Protein Res. 43, 431–440 (1994).
Johnson, T., Quibell, M. & Sheppard, R. C. N,O-bisFmoc derivatives of N-(2-hydroxy-4-methoxybenzyl)-amino acids: useful intermediates in peptide synthesis. J. Pept. Sci. 1, 11–25 (1995).
Zheng, J. S. et al. Robust chemical synthesis of membrane proteins through a general method of removable backbone modification. J. Am. Chem. Soc. 138, 3553–3561 (2016).
Jacobsen, M. T. et al. A helping hand to overcome solubility challenges in chemical protein synthesis. J. Am. Chem. Soc. 138, 11775–11782 (2016).
Wöhr, T. et al. Pseudo-prolines as a solubilizing, structure-disrupting protection technique in peptide synthesis. J. Am. Chem. Soc. 118, 9218–9227 (1996).
Pascal Dumy, M. K., Ryan, D. E., Rohwedder, B., Wöhr, T. & Mutter, M. Pseudo-prolines as a molecular hinge: reversible induction of cis amide bonds into peptide backbones. J. Am. Chem. Soc. 119, 918–925 (1997).
Sohma, Y. et al. ‘O-Acyl isopeptide method’ for the efficient synthesis of difficult sequence-containing peptides: use of ‘O-acyl isodipeptide unit’. Tetrahedron Lett. 47, 3013–3017 (2006).
Coin, I. The depsipeptide method for solid-phase synthesis of difficult peptides. J. Pept. Sci. 16, 223–230 (2010).
Kyte, J. & Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132 (1982).
Ellington, A. & Cherry, J. M. Characteristics of amino acids. Curr. Protoc. Mol. Biol. 33, A.1C.1–A.1C.12 (2001).
Fang, G. M., Wang, J. X. & Liu, L. Convergent chemical synthesis of proteins by ligation of peptide hydrazides. Angew. Chem. Int. Ed. Engl. 51, 10347–10350 (2012).
Zheng, J. S., Tang, S., Qi, Y. K., Wang, Z. P. & Liu, L. Chemical synthesis of proteins using peptide hydrazides as thioester surrogates. Nat. Protoc. 8, 2483–2495 (2013).
Xiong, A. S. et al. A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res. 32, e98 (2004).
Liu, X. & Zhu, T. F. Sequencing mirror-image DNA chemically. Cell Chem. Biol. 25, 1151–1156 (2018).
Nakamaye, K. L., Gish, G., Eckstein, F. & Vosberg, H.-P. Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside α-thiotriphosphates. Nucleic Acids Res. 16, 9947–9959 (1988).
Gish, G. & Eckstein, F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science 240, 1520–1522 (1988).
Sanger, F., Nicklen, S. & Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA 74, 5463–5467 (1977).
Zhang, B. et al. Ligation of soluble but unreactive peptide segments in the chemical synthesis of Haemophilus influenzae DNA ligase. Angew. Chem. Int. Ed. Engl. 58, 12231–12237 (2019).
Weidmann, J., Schnolzer, M., Dawson, P. E. & Hoheisel, J. D. Copying life: synthesis of an enzymatically active mirror-image DNA-ligase made of D-amino acids. Cell Chem. Biol. 26, 645–651 (2019).
Tiessen, A., Perez-Rodriguez, P. & Delaye-Arredondo, L. J. Mathematical modeling and comparison of protein size distribution in different plant, animal, fungal and microbial species reveals a negative correlation between protein size and protein number, thus providing insight into the evolution of proteomes. BMC Res. Notes 5, 85 (2012).
Zhang, B. C. et al. Chemical synthesis of proteins containing 300 amino acids. Chem. Res. Chin. Univ. 36, 733–747 (2020).
Cozens, C., Pinheiro, V. B., Vaisman, A., Woodgate, R. & Holliger, P. A short adaptive path from DNA to RNA polymerases. Proc. Natl Acad. Sci. USA 109, 8067–8072 (2012).
Church, G. M., Gao, Y. & Kosuri, S. Next-generation digital information storage in DNA. Science 337, 1628 (2012).
Goldman, N. et al. Towards practical, high-capacity, low-maintenance information storage in synthesized DNA. Nature 494, 77–80 (2013).
Ceze, L., Nivala, J. & Strauss, K. Molecular digital data storage using DNA. Nat. Rev. Genet. 20, 456–466 (2019).
Matange, K., Tuck, J. M. & Keung, A. J. DNA stability: a central design consideration for DNA data storage systems. Nat. Commun. 12, 1358 (2021).
Paunescu, D., Fuhrer, R. & Grass, R. N. Protection and deprotection of DNA–high-temperature stability of nucleic acid barcodes for polymer labeling. Angew. Chem. Int. Ed. Engl. 52, 4269–4272 (2013).
Koch, J. et al. A DNA-of-things storage architecture to create materials with embedded memory. Nat. Biotechnol. 38, 39–43 (2020).
Wade, D. et al. All-D amino acid-containing channel-forming antibiotic peptides. Proc. Natl Acad. Sci. USA 87, 4761–4765 (1990).
Caton-Williams, J., Hoxhaj, R., Fiaz, B. & Huang, Z. Use of a novel 5′-regioselective phosphitylating reagent for one-pot synthesis of nucleoside 5′-triphosphates from unprotected nucleosides. Curr. Protoc. Nucleic Acid Chem. 52, 1.30.1–1.30.21 (2013).
Huang, Y.-C. et al. Facile synthesis of C-terminal peptide hydrazide and thioester of NY-ESO-1 (A39-A68) from an Fmoc-hydrazine 2-chlorotrityl chloride resin. Tetrahedron 70, 2951–2955 (2014).
Huang, Y. C. et al. Synthesis of l- and d-ubiquitin by one-pot ligation and metal-free desulfurization. Chemistry 22, 7623–7628 (2016).
Maity, S. K., Jbara, M., Laps, S. & Brik, A. Efficient palladium-assisted one-pot deprotection of (acetamidomethyl)cysteine following native chemical ligation and/or desulfurization to expedite chemical protein synthesis. Angew. Chem. Int. Ed. Engl. 55, 8108–8112 (2016).
Burley, S. K. & Petsko, G. A. Weakly polar interactions in proteins. Adv. Protein Chem. 39, 125–189 (1988).
Lundberg, K. S. et al. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1–6 (1991).
Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).
Segata, N. et al. Metagenomic microbial community profiling using unique clade-specific marker genes. Nat. Methods 9, 811–814 (2012).
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.
The authors have filed a provisional patent application related to this work.
Peer review information Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
a, Design of information-storing L-DNA segments. Caret, uppercase. b, Experimental procedures for mirror-image DNA information storage.
About this article
Cite this article
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
This article is cited by
Nature Biotechnology (2022)