A multiplex high-throughput screening platform for investigation of buffer/binding conditions for aptamers

Abstract

Aim: Aptamers are oligonucleotide sequences designed for specific binding to a variety of target molecules ranging from small organic molecules to proteins. They are alternatives to antibodies and are commonly used in sensoring applications for biomedical research and toxicology. Binding affinity and specificity of aptamers depend strongly on factors like temperature, pH, ionic strength or divalent cations like Ca2+ or Mg2+. However, large scale investigations of aptamers is laborious. Therefore, we have build a fully automatic platform to analyze aptamer binding properties. Methods: This technology is based on the VideoScan platform [1], an automatized imaging system allowing the multiparametric analysis of up to eighteen biomolecules on a planar microbead array. Aptamer target proteins and non target proteins (e.g. INF, TNF-, enterotoxin B, GST) as negative controls were coupled to dye/size encoded microbeads. Binding of the fluorescent labeled aptamers on the surface of these microbeads under the chosen conditions were evaluated with the VideoScan platform. In this study, we focused on aptamers for lactate dehydrogenase from plasmodium falciparum (PfLDH), thrombin and streptavidin. Results: Whereas the streptavidin aptamer was highly specific for its target streptavidin, the PfLDH aptamer showed slight unspecific binding to thrombin and the thrombin aptamer strong unspecific binding to PfLDH. The streptavidin buffer was used for a systematic analysis of the influence of Ca-, Mg- and K-ions. We found that the SA-aptamer was strictly Ca- and Mg-dependent. In contrast, the PfLDH aptamer did not require these ions, the presence of Ca- and Mg-ions even increased unspecific binding. The binding buffer of PfLDH was modified by changing the pH-value and the addition of DMSO, TMAC, PEG 8000 and Tween 20. We found that low pH-values (pH <5) leads to completely unspecific binding. Our data suggest that 20% PEG 8000 and also 1% Tween 20 increased both, the specific and also unspecific binding up to 3 fold. While 5% DMSO had no effect, 3M TMAC prevents signal formation totally. A conditioning of aptamers by a rapid boiling/chilling cycle in the buffer had hardly any effect. We also compared the binding of thrombin aptamer with a thrombin antibody and found high correlation. Conclusion: Our technology is a promising tool for systematic analysis of aptamer properties. Due to the multiplexing capabilities it is possible to analyze multiple target and aptamers simultaneously. In future work we plan to use complex media (e. g., serum) to screen aptamers for their behaviours in real-world samples. [1] S. Rödiger, P. Schierack, A. Böhm, J. Nitschke, I. Berger, U. Frömmel, et al., A highly versatile microscope imaging technology platform for the multiplex real-time detection of biomolecules and autoimmune antibodies, Adv. Biochem. Eng. Biotechnol. 133 (2013) 35–74. doi:10.1007/10_2011_132.