A new paper (link is external) published this week in the Royal Society of Chemistry’s journal Chemical Communications showcases Affimer technology as applied to native and non-native protein interactions, demonstrating for the first time that the natural protein structure of Affimer binders can specifically interact with aromatic oligoamide proteomimetic foldamers.
The work of Professor Andrew Wilson’s team at the University of Leeds’ School of Chemistry used Affimer proteins in their analysis of foldamers. Foldamers are sequences of non-native monomers that can mimic the ability of proteins to fold into defined conformations, and have potential applications in areas such as inhibiting protein-protein interactions. The researchers identified Affimer binders to a series of foldamers constructed from N-alkylated aromatic oligoamide trimers, that were designed to mimic an ?-helix secondary structure and inhibit ?-helix mediated protein-protein interactions.
Probing the 3 x 1010 sequence strong Affimer library with six different biotinylated foldamer sequences, the Leeds University researchers were able to identify specific binders to four of these sequences, as determined by ELISA assay. This offers the first demonstration of the ability of native ?-amino acid protein structures to recognise aromatic oligoamide foldamers with high affinity and specificity, and highlights the utility of library screening techniques, such as Affimer technology, for the generation of affinity molecules to small molecules.
The authors of the study note that compatibility of the ‘natural’ protein and ‘non-natural’ proteomimetic foldamer codes in this way opens up a range of potential applications. Rapid library screening approaches could be used in the future to aid the discovery of natural biological ligands and targets of foldamers. Another application could be to identify macromolecules that could form non-covalent complexes with a foldamer, to be used as a starting point for development of hybrid macromolecule-foldamer tertiary structures.
To further query the interaction of the Affimer binders with the foldamers the team focussed on two specific foldamers from their series, determining the Affimer binders’ affinity for these molecules via titration by direct ELISA. Values of 4.8µM and 0.98µM demonstrate that the Affimer binders exhibit good affinity to the target foldamers, comparable to that of standard antibodies.
In competition assays with the biotinylated foldamers and untagged foldamers, the researchers were able to distinguish that while one of the Affimer binders showed no clear difference in binding to the biotinylated and non-biotinylated foldamer format, a second bound with a weak affinity to the untagged foldamer (>100µM) yet showed a high affinity to the biotinylated form (2.5µM). This variation in the binding affinity of the variously formatted foldamers was attributed to the Affimer binder interacting with the biotin tag of the foldamer in addition to the helix mimicking side chains of the foldamer. Employing a phage screening approach to select for a diversity of different binding modes in this way raises the potential for the technology to be used in elaborating complex 3D structures with natural and non-natural components.
The next phase of work that the research team plan to undertake involves screening phage libraries with foldamers in tandem with a bioinformatics approach to potentially identify disease relevant proteins as targets for foldamers.
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