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

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 Kd 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.

[1]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[2]  A. Pardi,et al.  A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF165. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[4]  Koji Sode,et al.  A screening method for DNA aptamers that bind to␣a␣specific, unidentified protein in tissue samples , 2006, Biotechnology Letters.

[5]  Sheela M. Waugh,et al.  2′-Fluoropyrimidine RNA-based Aptamers to the 165-Amino Acid Form of Vascular Endothelial Growth Factor (VEGF165) , 1998, The Journal of Biological Chemistry.

[6]  M. Nguyen.  Angiogenic factors as tumor markers , 2004, Investigational New Drugs.

[7]  Koji Sode,et al.  Novel electrochemical sensor system for protein using the aptamers in sandwich manner. , 2005, Biosensors & bioelectronics.

[8]  N. Ferrara,et al.  The biology of VEGF and its receptors , 2003, Nature Medicine.

[9]  H. Yoshida,et al.  Construction and characterization of mutant water-soluble PQQ glucose dehydrogenases with altered K(m) values--site-directed mutagenesis studies on the putative active site. , 1999, Biochemical and biophysical research communications.

[10]  B. Shen,et al.  Single-stranded DNA aptamers that bind differentiated but not parental cells: subtractive systematic evolution of ligands by exponential enrichment. , 2003, Journal of biotechnology.

[11]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[12]  R. Bhisitkul.  Vascular endothelial growth factor biology: clinical implications for ocular treatments , 2006, British Journal of Ophthalmology.

[13]  L. Gold,et al.  A tenascin-C aptamer identified by tumor cell SELEX: Systematic evolution of ligands by exponential enrichment , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Shay Soker,et al.  Neuropilin-1 Is Expressed by Endothelial and Tumor Cells as an Isoform-Specific Receptor for Vascular Endothelial Growth Factor , 1998, Cell.

[15]  Hung V. Nguyen,et al.  The Carboxyl-terminal Domain(111165) of Vascular Endothelial Growth Factor Is Critical for Its Mitogenic Potency (*) , 1996, The Journal of Biological Chemistry.

[16]  K. Ikebukuro,et al.  Aptameric enzyme subunit for biosensing based on enzymatic activity measurement. , 2006, Analytical chemistry.