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

Nature Biotechnology 31, 453–457 (2013) | Download Citation

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Abstract

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.

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References

  1. 1.

    , & Development of aptamer therapeutics. Curr. Opin. Pharmacol. 10, 557–562 (2010).

  2. 2.

    , & Aptamers: from bench side research towards patented molecules with therapeutic applications. Expert Opin. Ther. Pat. 19, 1603–1613 (2009).

  3. 3.

    , & Aptamers as therapeutics. Nat. Rev. Drug Discov. 9, 537–550 (2010).

  4. 4.

    & In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

  5. 5.

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

  6. 6.

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

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

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

  11. 11.

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

  12. 12.

    , , , & 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).

  13. 13.

    , , & Amplification, mutation, and sequencing of a six-letter synthetic genetic system. J. Am. Chem. Soc. 133, 15105–15112 (2011).

  14. 14.

    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).

  15. 15.

    , , , & An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules. Nucleic Acids Res. 37, e14 (2009).

  16. 16.

    , , , & Enzymatic incorporation of a third nucleobase pair. Nucleic Acids Res. 35, 4238–4249 (2007).

  17. 17.

    & 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).

  18. 18.

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

  19. 19.

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

  20. 20.

    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).

  21. 21.

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

  22. 22.

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

  23. 23.

    , , & Aptamer-based electrochemical biosensor for interferon gamma detection. Anal. Chem. 82, 8131–8136 (2010).

  24. 24.

    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).

  25. 25.

    & PCR product with strands of unequal length. Nucleic Acids Res. 23, 4220–4221 (1995).

  26. 26.

    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).

  27. 27.

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

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Acknowledgements

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.

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Affiliations

  1. RIKEN Systems and Structural Biology Center, Yokohama, Kanagawa, Japan.

    • Michiko Kimoto
    • , Rie Yamashige
    • , Ken-ichiro Matsunaga
    • , Shigeyuki Yokoyama
    •  & Ichiro Hirao

  2. TagCyx Biotechnologies, Yokohama, Kanagawa, Japan.

    • Michiko Kimoto
    •  & Ichiro Hirao

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Contributions

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.

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.

Corresponding author

Correspondence to Ichiro Hirao.

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DOI

https://doi.org/10.1038/nbt.2556