A Metal-free Click Chemistry Approach for the Assembly and Probing of Biomolecules.

Probing of biomolecular complexes by single-molecule force spectroscopy (SMFS) methods including AFM requires proper and suitable coupling methods for immobilization of biomolecules onto the AFM tip and the surface. The use of flexible tethers for the coupling process has dual advantages. First, they allow the specific immobilization of interacting molecules, and second, their flexibility facilitates the proper orientation of the interacting partners. Recently, we developed an approach termed Flexible Nano Array (FNA) in which interacting partners are located on the same polymeric FNA molecule separated by a flexible segment with a defined length. In this paper, we modified the FNA tether approach by incorporating click chemistry with non-metal modification. FNA was synthesized using DNA synthesis chemistry, in which phosphoramidite (PA) spacers containing six ethylene glycol units were used instead of nucleoside triphosphates. During the synthesis, two T modifiers conjugated to two dibenzocyclooctyl (DBCO) residues were incorporated at selected positions within the FNA. The DBCO functionality allows for coupling azide labeled biomolecules via click chemistry. Amyloid peptide Aβ(14-23) terminated with azide was incorporated into the FNA and the reaction was controlled with mass-spectrometry. Assembly of tethered Aβ(14-23) peptides into dimers was characterized by AFM force spectroscopy experiments in which the AFM tip functionalized with FNA terminated with biotin probed a streptavidin-coated mica surface. The formation of the peptide dimer was verified with force spectroscopy that showed the appearance of a specific fingerprint for dimer dissociation followed by a rupture event for the biotin-streptavidin link. The developed approach is capable of multiple probing events to allow the collection of a large set of data for a quantitative analysis of the force spectroscopy events.