Reversible bond formation enables the replication and amplification of a crosslinking salen complex as an orthogonal base pair


The universal genetic code relies on two hydrogen-bonded Watson–Crick base pairs that can form 64 triplet codons. This places a limit on the number of amino acids that can be encoded, which has motivated efforts to create synthetic base pairs that are orthogonal to the natural ones. An additional base pair would result in another 61 triplet codons. Artificial organic base pairs have been described in enzymatic incorporation studies, and inorganic T–Hg–T and C–Ag–C base pairs have been reported to form in primer extension studies. Here, we demonstrate a metal base pair that is fully orthogonal and can be replicated, and can even be amplified by polymerase chain reaction in the presence of the canonical pairs dA:dT and dG:dC. Crystal structures of a dS–Cu–dS base pair inside a polymerase show that reversible chemistry is possible directly inside the polymerase, which enables the efficient copying of the inorganic crosslink. The results open up the possibility of replicating and amplifying artificial inorganic DNA nanostructures by extending the genetic alphabet.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: An unnatural base pair system.
Figure 2: Primer extension experiments with the dS–dS base pair using Bst Pol I.
Figure 3: Crystal structures of modified DNA in Bst Pol I.
Figure 4: PCR amplification of multiple dS–Cu–dS base pairs.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank


  1. 1

    Giuseppone, N., Schmitt, J.-L. & Lehn, J.-M. Generation of dynamic constitutional diversity and driven evolution in helical molecular strands under Lewis acid catalyzed component exchange. Angew. Chem. Int. Ed. 43, 4902–4906 (2004).

    CAS  Article  Google Scholar 

  2. 2

    Lehn, J.-M. From supramolecular chemistry towards constitutional dynamic chemistry and adaptive chemistry. Chem. Soc. Rev. 36, 151–160 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Lu, Y. & Liu, J. Functional DNA nanotechnology: emerging applications of DNAzymes and aptamers. Curr. Opin. Biotechnol. 17, 580–588 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Niemeyer, C. M. Semisynthetic DNA–protein conjugates for biosensing and nanofabrication. Angew. Chem. Int. Ed. 49, 1200–1216 (2010).

    CAS  Article  Google Scholar 

  5. 5

    Seeman, N. C. DNA in a material world. Nature 421, 427–431 (2003).

    Article  Google Scholar 

  6. 6

    Switzer, C., Moroney, S. E. & Benner, S. A. Enzymatic incorporation of a new base pair into DNA and RNA. J. Am. Chem. Soc. 111, 8322–8323 (1989).

    CAS  Article  Google Scholar 

  7. 7

    Crick, F. H. C., Barnett, L., Brenner, S. & Watts-Tobin, R. J. General nature of the genetic code for proteins. Nature 192, 1227–1232 (1961).

    CAS  Article  Google Scholar 

  8. 8

    Kool, E. T. Synthetically modified DNAs as substrates for polymerases. Curr. Opin. Chem. Biol. 4, 602–608 (2000).

    CAS  Article  Google Scholar 

  9. 9

    Henry, A. A. & Romesberg, F. E. Beyond A, C, G and T: augmenting Nature's alphabet. Curr. Opin. Chem. Biol. 7, 727–733 (2003).

    CAS  Article  Google Scholar 

  10. 10

    Hirao, I. Unnatural base pair systems for DNA/RNA-based biotechnology. Curr. Opin. Chem. Biol. 10, 622–627 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Kool, E. T. Replacing the nucleobases in DNA with designer molecules. Acc. Chem. Res. 35, 936–943 (2002).

    CAS  Article  Google Scholar 

  12. 12

    Kool, E. T., Morales, J. C. & Guckian, K. M. Mimicking the structure and function of DNA: insights into DNA stability and replication. Angew. Chem. Int. Ed. 39, 990–1009 (2000).

    CAS  Article  Google Scholar 

  13. 13

    Krueger, A. T. & Kool, E. T. Redesigning the architecture of the base pair: toward biochemical and biological function of new genetic sets. Chem. Biol. 16, 242–248 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Switzer, C. Y., Moroney, S. E. & Benner, S. A. Enzymic recognition of the base pair between isocytidine and isoguanosine. Biochemistry 32, 10489–10496 (1993).

    CAS  Article  Google Scholar 

  15. 15

    Killelea, T. et al. Probing the interaction of archaeal DNA polymerases with deaminated bases using X-ray crystallography and non-hydrogen bonding isosteric base analogues. Biochemistry 49, 5772–5781 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Kimoto, M., Mitsui, T., Yokoyama, S. & Hirao, I. A unique fluorescent base analogue for the expansion of the genetic alphabet. J. Am. Chem. Soc. 132, 4988–4989 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Leconte, A. M. et al. Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet. J. Am. Chem. Soc. 130, 2336–2343 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Lu, H., Krueger, A. T., Gao, J., Liu, H. & Kool, E. T. Toward a designed genetic system with biochemical function: polymerase synthesis of single and multiple size-expanded DNA base pairs. Org. Biomol. Chem. 8, 2704–2710 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Matsuda, S. et al. Efforts toward expansion of the genetic alphabet: structure and replication of unnatural base pairs. J. Am. Chem. Soc. 129, 10466–10473 (2007).

    CAS  Article  Google Scholar 

  20. 20

    Moran, S., Ren, R. X. F. & Kool, E. T. A thymidine triphosphate shape analog lacking Watson–Crick pairing ability is replicated with high sequence selectivity. Proc. Natl Acad. Sci. USA 94, 10506–10511 (1997).

    CAS  Article  Google Scholar 

  21. 21

    Moran, S., Ren, R. X. F., Rumney, S. I. V. & Kool, E. T. Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA replication. J. Am. Chem. Soc. 119, 2056–2057 (1997).

    CAS  Article  Google Scholar 

  22. 22

    Hirao, I. et al. An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA. Nature Methods 3, 729–735 (2006).

    CAS  Article  Google Scholar 

  23. 23

    Hirao, I., Mitsui, T., Kimoto, M. & Yokoyama, S. Development of an unnatural base pair for efficient PCR amplification. Nucleic Acids Symp. Ser. 51, 9–10 (2007).

    Article  Google Scholar 

  24. 24

    Johnson, S. C., Sherrill, C. B., Marshall, D. J., Moser, M. J. & Prudent, J. R. A third base pair for the polymerase chain reaction: inserting isoC and isoG. Nucleic Acids Res. 32, 1937–1941 (2004).

    CAS  Article  Google Scholar 

  25. 25

    Yang, Z., Chen, F., Chamberlin, S. G. & Benner, S. A. Expanded genetic alphabets in the polymerase chain reaction. Angew. Chem. Int. Ed. 49, 177–180 (2010).

    CAS  Article  Google Scholar 

  26. 26

    Piccirilli, J. A., Krauch, T., Moroney, S. E. & Benner, S. A. Enzymic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature 343, 33–37 (1990).

    CAS  Article  Google Scholar 

  27. 27

    Lutz, M. J., Horlacher, J. & Benner, S. A. Recognition of a non-standard base pair by thermostable DNA polymerases. Bioorg. Med. Chem. Lett. 8, 1149–1152 (1998).

    CAS  Article  Google Scholar 

  28. 28

    Urata, H., Yamaguchi, E., Funai, T., Matsumura, Y. & Wada, S.-i. Incorporation of thymine nucleotides by DNA polymerases through T-HgII-T base pairing. Angew. Chem. Int. Ed. 49, 6516–6519 (2010).

    CAS  Article  Google Scholar 

  29. 29

    Katz, S. The reversible reaction of sodium thymonucleate and mercuric chloride. J. Am. Chem. Soc. 74, 2238–2245 (1952).

    CAS  Article  Google Scholar 

  30. 30

    Kuklenyik, Z. & Marzilli, L. G. Mercury(II) site-selective binding to a DNA hairpin. Relationship of sequence-dependent intra- and interstrand cross-linking to the hairpin–duplex conformational transition. Inorg. Chem. 35, 5654–5662 (1996).

    CAS  Article  Google Scholar 

  31. 31

    Miyake, Y. et al. MercuryII-mediated formation of thymine–HgII–thymine base pairs in DNA duplexes. J. Am. Chem. Soc. 128, 2172–2173 (2006).

    CAS  Article  Google Scholar 

  32. 32

    Park, K. S., Jung, C. & Park, H. G. ‘Illusionary’ polymerase activity triggered by metal ions: use for molecular logic-gate operations. Angew. Chem. Int. Ed. 49, 9757–9760 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Ono, A. et al. Specific interactions between silver(I) ions and cytosine–cytosine pairs in DNA duplexes. Chem. Commun. 39, 4825–4827 (2008).

    Article  Google Scholar 

  34. 34

    Urata, H., Yamaguchi, E., Nakamura, Y. & Wada, S-i. Pyrimidine–pyrimidine base pairs stabilized by silver(I) ions. Chem. Commun. 47, 941–943 (2011).

    CAS  Article  Google Scholar 

  35. 35

    Carell, T. Molecular computing: DNA as a logic operator. Nature 469, 45–46 (2011).

    CAS  Article  Google Scholar 

  36. 36

    Czlapinski, J. L. & Sheppard, T. L. Nucleic acid template-directed assembly of metallosalen–DNA conjugates. J. Am. Chem. Soc. 123, 8618–8619 (2001).

    CAS  Article  Google Scholar 

  37. 37

    Czlapinski, J. L. & Sheppard, T. L. Template-directed assembly of metallosalen–DNA hairpin conjugates. ChemBioChem 5, 127–129 (2004).

    CAS  Article  Google Scholar 

  38. 38

    Gothelf, K. V., Thomsen, A., Nielsen, M., Cló, E. & Brown, R. S. Modular DNA-programmed assembly of linear and branched conjugated nanostructures. J. Am. Chem. Soc. 126, 1044–1046 (2004).

    CAS  Article  Google Scholar 

  39. 39

    Nielsen, M., Thomsen, A. H., Cló, E., Kirpekar, F. & Gothelf, K. V. Synthesis of linear and tripoidal oligo(phenylene ethynylene)-based building blocks for application in modular DNA-programmed assembly. J. Org. Chem. 69, 2240–2250 (2004).

    CAS  Article  Google Scholar 

  40. 40

    Clever, G. H., Polborn, K. & Carell, T. A highly DNA-duplex-stabilizing metal-salen base pair. Angew. Chem. Int. Ed. 44, 7204–7208 (2005).

    CAS  Article  Google Scholar 

  41. 41

    Clever, G. H. & Carell, T. Controlled stacking of 10 transition-metal ions inside a DNA duplex. Angew. Chem. Int. Ed. 46, 250–253 (2007).

    CAS  Article  Google Scholar 

  42. 42

    Clever, G. H., Reitmeier, S. J., Carell, T. & Schiemann, O. Antiferromagnetic coupling of stacked cuII-salen complexes in DNA. Angew. Chem. Int. Ed. 49, 4927–4929 (2010).

    CAS  Article  Google Scholar 

  43. 43

    Clever, G. H., Soeltl, Y., Burks, H., Spahl, W. & Carell, T. Metal–salen-base-pair complexes inside DNA: complexation overrides sequence information. Chem. Eur. J. 12, 8708–8718 (2006).

    CAS  Article  Google Scholar 

  44. 44

    Clever, G. H., Kaul, C. & Carell, T. DNA–metal base pairs. Angew. Chem. Int. Ed. 46, 6226–6236 (2007).

    CAS  Article  Google Scholar 

  45. 45

    Müller, J. Metal-ion-mediated base pairs in nucleic acids. Eur. J. Inorg. Chem. 3749–3763 (2008).

    Article  Google Scholar 

  46. 46

    Clever, G. H. & Shionoya, M. Metal–base pairing in DNA. Coord. Chem. Rev. 254, 2391–2402 (2010).

    CAS  Article  Google Scholar 

  47. 47

    Yoon, T. P. & Jacobsen, E. N. Privileged chiral catalysts. Science 299, 1691–1693 (2003).

    CAS  Article  Google Scholar 

  48. 48

    Atwell, S., Meggers, E., Spraggon, G. & Schultz, P. G. Structure of a copper-mediated base pair in DNA. J. Am. Chem. Soc. 123, 12364–12367 (2001).

    CAS  Article  Google Scholar 

  49. 49

    Johannsen, S., Megger, N., Boehme, D., Sigel, R. K. O. & Mueller, J. Solution structure of a DNA double helix with consecutive metal-mediated base pairs. Nature Chem. 2, 229–234 (2010).

    CAS  Article  Google Scholar 

  50. 50

    Johannsen, S., Paulus, S., Düpre, N., Müller, J. & Sigel, R. K. O. Using in vitro transcription to construct scaffolds for one-dimensional arrays of mercuric ions. J. Inorg. Biochem. 102, 1141–1151 (2008).

    CAS  Article  Google Scholar 

Download references


The authors acknowledge financial support from the Deutsche Forschungsgemeinschaft (SFB749, CiPSM) and the Volkswagen Foundation (grant no. I/85052). C.K. is the recipient of a pre-doctoral fellowship from the Fonds der Chemischen Industrie.

Author information




C.K. and M.W. performed the chemical experiments. C.K. carried out the biochemical assays and crystallization experiments. M.M. solved the crystal structures and supervised the biological studies. S.S. collected crystal data. T.C. designed the study and supervised the research project. C.K., M.M. and T.C. wrote the manuscript.

Corresponding author

Correspondence to Thomas Carell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 1255 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kaul, C., Müller, M., Wagner, M. et al. Reversible bond formation enables the replication and amplification of a crosslinking salen complex as an orthogonal base pair. Nature Chem 3, 794–800 (2011).

Download citation

Further reading