Application Notes

Analyzing the binding affinity of aptamer quantum dot conjugates to VEGF

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2 target-binding sites simultaneously interact with it. On the other hand the aptamers are very close to each other which may cause steric hindrance. The aim of this study was to use MicroScale Thermophoresis (Jerabek-Willemsen M. et al., 2011) in order to characterize the binding of fluorescently labeled VEGF protein to Quantum dots, which were conjugated with DNA aptamers. Results Here, we investigated the binding of NT-647- labeled VEGF to quantum dots, which were conjugated with the VEGF binding aptamer V7t1 (Nonaka Y. et al. 2010) via a 3´terminal amino linker fused to the aptamer. Fig. 2: Binding of NT-647-labeled VEGF to quantum dots – V7t1 3'. In the MST experiment, we kept the concentration of fluorescently-labeled VEGF constant at 20 nM, while theconcentration of the quantum dots V7t1 3' was varied. After 10 min, an MST analysis was performed (n = 3). A Kd of 27 nM ± 5.3 nM was determined for this interaction The concentration of NT-647 labeled VEGF was kept constant at 20 nM, while the concentration of the quantum dots was varied. After a short incubation, the samples were loaded into MST hydrophilic glass capillaries (K004, NanoTemper Technologies GmbH, Germany) and MST analysis was performed. The calculated K d for the interaction between NT-647-labeled VEGF and the V7t1 aptamer-conjugated quantum dots was 27 nM ± 5 nM. We observed, as expected, no binding of fluorescently labeled BSA (Bovine Serum Albumin) to the aptamer conjugated quantum dots (Fig. 2). Material and Methods Assay conditions Binding experiments were carried out in aptamer selection buffer supplemented with KCl (10 mM Tris/HCl, 100 mM NaCl, 0.05 mM EDTA, 50 mM KCl, pH 7.0). For the experiment VEGF protein was labeled with the Monolith NT TM Protein labeling Kit BLUE according to the supplied labeling protocol. Labeled VEGF was used at a concentration of 20 nM. The quantum dots were titrated in 1:1 dilution beginning at 200 nM. Monolith NT.115 hydrophilic capillaries were used in all experiments. Instrumentation The measurements were conducted on a NanoTemper Monolith.NT115 instrument. The Analysis was performed at 50 % LED power and 20 % MST power. "Fluo. Before" time was 5 seconds. "MST on time" was 30 seconds and "Fluo. After" time was 5 seconds. Conclusion This study provides another example that MicroScale Thermophoresis is capable of measuring interactions of biomolecules with larger particles such as nanoparticles or quantum dots. References Finetti F. et al., Functional and pharmacological characterization of a VEGF mimetic peptide on reparative angiogenesis. Biochemical Pharmacology (2012) 84:303–11. You K.M. et al., Aptamers as functional nucleic acids:In vitroselection and biotechnological applications. Biotechnol Bioproc (2012)E 8:64–75. Nonaka Y. et al., Screening and Improvement of an Anti- VEGF DNA Aptamer. Molecules (2010) 15 (1): 215-225. Walter J.-G. et al., Aptasensors for Small Molecule Detection. Z. Naturforsch.(2012) B 67:976–86. Walter J.-G. et al., Systematic Investigation of Optimal Aptamer Immobilization for Protein−Microarray Applications Anal. Chem. (2008) 80:7372–8. Nonaka Y. et al., Screening and Improvement of an Anti- VEGF DNA Aptamer. Molecules (2010) 15:215–25. Zhou D., Quantum dot–nucleic acid/aptamer bioconjugate- based fluorimetric biosensors. Biochem. Soc. Trans.(2012) 40:635–9. Jerabek-Willemsen M. et. al, Molecular Interaction studies using microscale Thermophoresis. Assay Drug Dev Technol. Aug;9(4):342-53(2011). © 2013 NanoTemper Technologies GmbH

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