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

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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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).

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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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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.

Corresponding author

Correspondence to Ichiro Hirao.

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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.

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

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

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