Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Generation of high-affinity DNA aptamers using an expanded genetic alphabet


DNA aptamers produced with natural or modified natural nucleotides often lack the desired binding affinity and specificity to target proteins. Here we describe a method for selecting DNA aptamers containing the four natural nucleotides and an unnatural nucleotide with the hydrophobic base 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). We incorporated up to three Ds nucleotides in a random sequence library, which is expected to increase the chemical and structural diversity of the DNA molecules. Selection experiments against two human target proteins, vascular endothelial cell growth factor-165 (VEGF-165) and interferon-γ (IFN-γ), yielded DNA aptamers that bind with KD values of 0.65 pM and 0.038 nM, respectively, affinities that are >100-fold improved over those of aptamers containing only natural bases. These results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: DNA aptamer selection using libraries containing hydrophobic Ds bases.
Figure 2: Characterizations and binding affinities of anti–VEGF-165 aptamer (VGd1-2Ds-47) and anti–IFN-γ aptamer (IFd1-3Ds-49).


  1. Bunka, D.H.J., Platonova, O. & Stockley, P.G. Development of aptamer therapeutics. Curr. Opin. Pharmacol. 10, 557–562 (2010).

    Article  CAS  PubMed  Google Scholar 

  2. Majumder, P., Gomes, K.N. & Ulrich, H. Aptamers: from bench side research towards patented molecules with therapeutic applications. Expert Opin. Ther. Pat. 19, 1603–1613 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Keefe, A.D., Pai, S. & Ellington, A. Aptamers as therapeutics. Nat. Rev. Drug Discov. 9, 537–550 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ellington, A.D. & Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    Article  CAS  PubMed  Google Scholar 

  5. Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).

    Article  CAS  PubMed  Google Scholar 

  6. Rich, A. in Horizons in Biochemistry (eds., M. Kasha & B. Pullman) 103–126 (Academic Press, 1962).

  7. Gold, L. et al. Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS ONE 5, e15004 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Vater, A. & Klussmann, S. Toward third-generation aptamers: Spiegelmers and their therapeutic prospects. Curr. Opin. Drug Discov. Devel. 6, 253–261 (2003).

    CAS  PubMed  Google Scholar 

  9. Campbell, M.A. & Wengel, J. Locked vs. unlocked nucleic acids (LNA vs. UNA): contrasting structures work towards common therapeutic goals. Chem. Soc. Rev. 40, 5680–5689 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. Pinheiro, V.B. et al. Synthetic genetic polymers capable of heredity and evolution. Science 336, 341–344 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yamashige, R. et al. Highly specific unnatural base pair systems as a third base pair for PCR amplification. Nucleic Acids Res. 40, 2793–2806 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Seo, Y.J., Malyshev, D.A., Lavergne, T., Ordoukhanian, P. & Romesberg, F.E. Site-specific labeling of DNA and RNA using an efficiently replicated and transcribed class of unnatural base pairs. J. Am. Chem. Soc. 133, 19878–19888 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yang, Z., Chen, F., Alvarado, J.B. & Benner, S.A. Amplification, mutation, and sequencing of a six-letter synthetic genetic system. J. Am. Chem. Soc. 133, 15105–15112 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Malyshev, D.A. et al. Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet. Proc. Natl. Acad. Sci. USA 109, 12005–12010 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kimoto, M., Kawai, R., Mitsui, T., Yokoyama, S. & Hirao, I. An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules. Nucleic Acids Res. 37, e14 (2009).

    Article  PubMed  Google Scholar 

  16. Yang, Z., Sismour, A.M., Sheng, P., Puskar, N.L. & Benner, S.A. Enzymatic incorporation of a third nucleobase pair. Nucleic Acids Res. 35, 4238–4249 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hirao, I. & Kimoto, M. Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma. Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 88, 345–367 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. Potty, A.S.R. et al. Biophysical characterization of DNA aptamer interactions with vascular endothelial growth factor. Biopolymers 91, 145–156 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Ruckman, J. et al. 2′-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J. Biol. Chem. 273, 20556–20567 (1998).

    Article  CAS  PubMed  Google Scholar 

  21. Ng, E.W. et al. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat. Rev. Drug Discov. 5, 123–132 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Xiang, Y. & Lu, Y. Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nat. Chem. 3, 697–703 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Liu, Y., Tuleouva, N., Ramanculov, E. & Revzin, A. Aptamer-based electrochemical biosensor for interferon gamma detection. Anal. Chem. 82, 8131–8136 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Tam, S. et al. Suppression of interferon-gamma induction of MHC class II and ICAM-1 by a 26-base oligonucleotide composed of deoxyguanosine and deoxythymidine. Transpl. Immunol. 2, 285–292 (1994).

    Article  CAS  PubMed  Google Scholar 

  25. Williams, K.P. & Bartel, D.P. PCR product with strands of unequal length. Nucleic Acids Res. 23, 4220–4221 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hirao, I. et al. In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA. Biochemistry 43, 3214–3221 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Tuleuova, N. et al. Development of an aptamer beacon for detection of interferon-gamma. Anal. Chem. 82, 1851–1857 (2010).

    Article  CAS  PubMed  Google Scholar 

Download references


We thank F.E. Romesberg and B. Hodošček for stimulating discussions and A. Sato for chemical synthesis. This work was supported by Grants-in-Aid for Scientific Research (KAKENHI 19201046 to I.H.) and by the Targeted Proteins Research Program and the RIKEN Structural Genomics/Proteomics Initiative, the National Project on Protein Structural and Functional Analyses, from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Author information

Authors and Affiliations



M.K. and I.H. conceived the project, designed methods and experiments and wrote the manuscript; R.Y. and K.M. performed in vitro selection targeting VEGF-165 and IFN-γ, respectively; M.K., R.Y. and K.M. analyzed the aptamers' binding and structural properties; M.K., R.Y., K.M. and I.H. jointly analyzed the data sets; I.H. and S.Y. supervised the project.

Corresponding author

Correspondence to Ichiro Hirao.

Ethics declarations

Competing interests

A patent application describing ideas presented in this paper has been filed by TagCyx Biotechnologies and RIKEN (PCT/JP2012/079611 by I.H., M.K., R.Y. and S.Y.). M.K. and I.H. own stock in TagCyx Biotechnologies.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–9, Supplementary Methods (PDF 2863 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kimoto, M., Yamashige, R., Matsunaga, Ki. et al. Generation of high-affinity DNA aptamers using an expanded genetic alphabet. Nat Biotechnol 31, 453–457 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing