Biotechnology Letters

, Volume 28, Issue 23, pp 1933–1937 | Cite as

Analysis of the evolution of the thrombin-inhibiting DNA aptamers using a genetic algorithm

  • Kazunori IkebukuroEmail author
  • Wataru Yoshida
  • Takahisa Noma
  • Koji Sode
Original Research Paper


We previously identified a thrombin-inhibiting DNA aptamer that was presumed to form a G-quartet structure with a duplex. To investigate the importance of the sequences in the duplex region and to obtain aptamers with higher inhibitory activities, we randomized the sequences of the duplex region of this aptamer and carried out selection based on inhibitory activity using a genetic algorithm. This method consisted of selection via an inhibition assay, crossover, and mutation in silico. After two cycles, we obtained ligands with greater inhibitory activities than that of the original aptamer. In addition, the duplex sequences were found to contribute to the inhibitory activities of aptamers.


Aptamer Genetic algorithm G-quartet structure Inhibitor Systematic evolution of ligands by exponential enrichment 



This work is partly supported by the Grant-in-Aid for the 21st Century COE “Future Nano-Materials” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.


  1. Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ (1992) Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355:564–566PubMedCrossRefGoogle Scholar
  2. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822PubMedCrossRefGoogle Scholar
  3. Ikebukuro K, Okumura Y, Sumikura K, Karube I (2005) A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm. Nucleic Acids Res 33:e108PubMedCrossRefGoogle Scholar
  4. Noma T, Ikebukuro K (2006) Aptamer selection based on inhibitory activity using an evolution-mimicking algorithm. Biochem Biophys Res Commun (in press). DOI 10.1016/j.bbrc.2006.06.083Google Scholar
  5. Padmanabhan K, Padmanabhan KP, Ferrara JD, Sadler JE, Tulinsky A (1993) The structure of alpha-thrombin inhibited by a 15-mer single-stranded DNA aptamer. J Biol Chem 268:17651–17654PubMedGoogle Scholar
  6. Ruckman J, Green LS, Beeson J, Waugh S, Gillette WL, Henninger DD, Claesson-Welsh L, Janjic N (1998) 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 273:20556–20567PubMedCrossRefGoogle Scholar
  7. Singh J, Ator MA, Jaeger EP, Allen MP, Whipple DA, Soloweij JE, Chowdhary S, Treasurywala AM (1996) Application of genetic algorithms to combinatorial synthesis: A computational approach to lead identification and lead optimization. J Am Chem Soc 118:1669–1676CrossRefGoogle Scholar
  8. Tasset DM, Kubik MF, Steiner W (1997) Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. J Mol Biol 272:688–698PubMedCrossRefGoogle Scholar
  9. Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510PubMedCrossRefGoogle Scholar
  10. Weber L, Wallbaum S, Broger C, Gubernator K (1995) A genetic algorithm optimizing the biological activity of combinatorial compound libraries. Angew Chem Int Ed Engl 107:2453–2454Google Scholar
  11. Yokobayashi Y, Ikebukuro K, McNiven S, Karube I (1996) Directed evolution of trypsin inhibiting peptides using a genetic algorithm. J Chem Soc Perkin Trans I 20:2435–2437CrossRefGoogle Scholar
  12. Zhang W, Loughran MG, Kanna S, Yano K, Ikebukuro K, Yokobayashi Y, Kuroda R, Karube I (2003) Exploration of structural features of monomeric helical peptides designed with a genetic algorithm. Proteins 53:193–200PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Kazunori Ikebukuro
    • 1
    Email author
  • Wataru Yoshida
    • 1
  • Takahisa Noma
    • 1
  • Koji Sode
    • 1
  1. 1.Department of Life Science and BiotechnologyTokyo University of Agriculture and TechnologyKoganei, TokyoJapan

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