Skip to main content
SpringerLink
Log in

Selection of DNA aptamers against VEGF165 using a protein competitor and the aptamer blotting method

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

Two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF165) were identified. In the screening process, another protein was used as the competitor to isolate those aptamers that have high specificity for the target. In addition, we evaluated the affinities of the enriched library by means of aptamer blotting. The isolated aptamers bound to VEGF165 with a K d value in the range of a few hundred nanomoles, and did not bind to the competitor. This selection method enabled us to efficiently select the specific aptamers against the target protein. These specific aptamers would be useful sensor elements for cancer diagnosis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Bhisitkul RB (2006) Vascular endothelial growth factor biology: clinical implications for ocular treatments. Br J Ophthalmol 90:1542–1547

    Article  PubMed  CAS  Google Scholar 

  • Daniels DA, Chen H, Hicke BJ, Swiderek KM, Gold L (2003) A tenascin-C aptamer identified by tumor cell SELEX: systematic evolution of ligands by exponential enrichment. Proc Natl Acad Sci USA 100:15416–15421

    Article  PubMed  CAS  Google Scholar 

  • Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822

    Article  PubMed  CAS  Google Scholar 

  • Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nat Med 9:669–676

    Article  PubMed  CAS  Google Scholar 

  • Igarashi S, Ohtera T, Yoshida H, Witarto AB, Sode K (1999) Construction and characterization of mutant water-soluble PQQ glucose dehydrogenases with altered K(m) values-site-directed mutagenesis studies on the putative active site. Biochem Biophys Res Commun 26:820–824

    Article  Google Scholar 

  • Ikebukuro K, Kiyohara C, Sode K (2005) Novel electrochemical sensor system for protein using the aptamers in sandwich manner. Biosens Bioelectron 20:2168–2172

    Article  PubMed  CAS  Google Scholar 

  • Keyt BA, Berleau LT, Nguyen HV, Chen H, Heinsohn H, Vandlen R, Ferrara N (1996) The carboxyl-terminal domain (111–165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem 271:7788–7795

    Article  PubMed  CAS  Google Scholar 

  • Lee JH, Canny MD, De Erkenez A, Krilleke D, Ng YS, Shima DT, Pardi A, Jucker F (2005) A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF165. Proc Natl Acad Sci USA 102:18902–18907

    Article  PubMed  CAS  Google Scholar 

  • Mathews DH, Sabina J, Zuker M, Turner DH (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288:911–940

    Article  PubMed  CAS  Google Scholar 

  • Nguyen M (1997) Angiogenic factors as tumor markers. Invest New Drugs 15:29–37

    Article  PubMed  CAS  Google Scholar 

  • Noma T, Ikebukuro K, Sode K, Ohkubo T, Sakasegawa Y, Hachiya N, Kaneko K (2006) A screening method for DNA aptamers that bind to a specific, unidentified protein in tissue samles. Biotechnol Lett 28:1377–1381

    Article  PubMed  CAS  Google Scholar 

  • Ruckman J, Green LS, Beeson J, Waugh S, Gillette WL, Henninger DD, Claesson-Welsh L, Janjić N (1998) 2′-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). J Biol Chem 273:20556–20567

    Article  PubMed  CAS  Google Scholar 

  • Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M (1998) Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92:735–745

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Wang C, Zhang M, Yang G, Zhang D, Ding H, Wang H, Fan M, Shen B, Shao N (2003) Single-stranded DNA aptamers that bind differentiated but not parental cells: subtractive systematic evolution of ligands by exponential enrichment. J Biotechnol 102:15–22

    Article  PubMed  CAS  Google Scholar 

  • Yoshida W, Sode K, Ikebukuro K (2006) Aptameric enzyme subunit for biosensing based on enzymatic activity measurement. Anal Chem 78:3296–3303

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the 2006 Industrial Technology Research Grant Program of the New Energy and Industrial Technology Development Organization (NEDO) of Japan.

Author information

Authors and Affiliations

  1. Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan

    Hijiri Hasegawa, Koji Sode & Kazunori Ikebukuro

Authors
  1. Hijiri Hasegawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Koji Sode
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kazunori Ikebukuro
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kazunori Ikebukuro.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hasegawa, H., Sode, K. & Ikebukuro, K. Selection of DNA aptamers against VEGF165 using a protein competitor and the aptamer blotting method. Biotechnol Lett 30, 829–834 (2008). https://doi.org/10.1007/s10529-007-9629-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-007-9629-6

Keywords

Search

Navigation