Abstract: Manuel MAIDORN

Novel affinity probes enhance resolution in high-resolution microscopy

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Manuel Maidorn, Felipe Opazo

UMG, Götingen, Germany.

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During the last decades, high-resolution microscopy techniques such as Stimulated Emission Depletion (STED) or Stochastic Optical Reconstruction Microscopy (STORM) improved resolution of biological structures to the low nanometer range. The detection of subcellular structures by fluorescence microscopy largely relies on assays based on affinity probes selectively binding to particular target molecules. However, it has been noticed that the large size of conventional affinity probes, such as immunoglobulins (~150 kDa), impairs accuracy of biological stainings. In particular, this can be observed in super-resolution microscopy. Consequently, the development of smaller molecular probes is mandatory to exploit the full potential of today’s super-resolution instruments. An alternative to bulky immunoglobulin molecules are single domain antibodies obtained from camelids such as llama and alpaca. Those probes, called nanobodies, show several advantages over conventional immunoglobulins. Their spatial size ranges around 3 nm with a total molecular weight of 15 kDa. They are strictly monovalent and mainly characterized by their high molecular stability and solubility. Nanobodies with high and specific binding capability can be selected from comprehensive immune libraries, using phage display technology. We will select and optimize such novel probes for super-resolution microscopy. Due to a controlled site-directed coupling of nanobodies to fluorescent molecules, we can obtain monovalent probes possessing a single fluorophore. This will provide a basis for quantitative studies of endogenous protein levels bypassing the need for genetic modifications.