Application Notes

Analyzing the binding affinity of aptamer quantum dot conjugates to VEGF

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1 Protein-Quantum Dots Interaction Analysis Application Note NT-MO-019 Analyzing the binding affinity of aptamer quantum dot conjugates to VEGF Moran Jerabek-Willemsen 1 , Maren Lönne 2 , Johanna-Gabriela Walter 2 , Thomas Scheper 2 , Stefan Duhr 1 1 NanoTemper Technologies GmbH, Munich, Germany 2 Institut für Technische Chemie, Leibniz Universität Hannover, Germany Abstract Aptamers are single-stranded oligonucleotides which fold into a three-dimensional structure allowing the specific recognition of a molecular target. Therefore, they might be an alternative to antibodies as detection molecules in biosensors. Aptamers are well suited for this application since they can be directed against almost every molecular target, and show high binding affinities to their corresponding binding partner. Aptamers with a terminal amino-group can be coupled to organic or inorganic fluorophores via amide bonds to enable their optical detection. In this study Vascular Endothelial Growth Factor (VEGF)-binding aptamers were conjugated with quantum dot molecules. This immobilization may inhibit the correct aptamer folding and consequently lead to a declined target-binding activity. Therefore, MST technology was used to determine whether quantum dot conjugation affects the VEGF- binding affinity of aptamers. Introduction Vascular Endothelial Growth Factor (VEGF) is a bioactive protein which is used as a supplement in cell culture media (Finetti F. et al., 2012). It is biotechnologically produced in microorganisms or mammalian cells and afterwards purified from the complex culture broth. Monitoring the purification process demands a specific and sensitive detection platform for VEGF. Aptamers fulfill these requirements as they bind to a single target with high affinity and specificity. Aptamers are single-stranded DNA or RNA molecules with length of ~15 – 60 nucleobases. Some of these bases interact with each other resulting in a particular aptamer folding. This three-dimensional molecular structure enables the aptamer to bind its corresponding target (You K.M. et al., 2012). Fig. 1: Schematic representation of VEGF binding to an aptamer-Qdot-conjugate During chemical synthesis a functional group as an amino group or a carboxyl group can be added to the 3' or 5' terminal mononucleotide of the aptamer. Conjugation with a corresponding carboxyl- or amino-functionalized fluorophore results in a fluorescence-labeled aptamer which can be used as detection molecule in optical biosensors (Walter J.-G. et al., 2012). However, due to steric hindrance or electrostatic interactions between aptamer and fluorophore the conjugation might interfere with the normal aptamer folding. Usually this results in a declined target-binding affinity (Walter J.-G. et al., 2008). In this study VEGF-binding aptamers were conjugated with quantum dot molecules, which are semiconductor nanocrystals that emit fluorescent light (Zhou D., 2012). They display multiple functional groups on their surface and therefore they bind more than one aptamer. On the one hand this might enlarge the strength of the VEGF binding since multiple

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