Abstract
Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.
Highlights
Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can fold into unique, three-dimensional shapes
Binding of AS1411-cotinine/anti-cotinine antibody complexes to cell-surface nucleolin
To assess whether AS1411-cotinine/anti-cotinine antibody complexes (Figures 1, 2) bind to nucleolin on cell surfaces, Raji cells were incubated with AS1411-cotinine/anti-cotinine antibody complexes and Fluorescein isothiocyanate (FITC)-labeled anti-human IgG antibodies
Summary
Relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can fold into unique, three-dimensional shapes. Aptamers have been used in a variety of methods in which antibodies are commonly used, such as in enzyme immunoassays, immunoprecipitation analyses, flow cytometric analyses (Ireson and Kelland, 2006; Ferreira et al, 2008; Sakai et al, 2008), protein microarrays (Chen et al, 2008), magnetic-separation assays (Gao et al, 2007), lateral flow assays (Liu et al, 2007; Shaikh et al, 2007), and biosensor experiments (Backmann et al, 2005; Borisov and Wolfbeis, 2008). Labeling of aptamers with biotin to produce complexes with avidin, streptavidin, or neutravidin
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