During the fellowship a robust microbead surface display technique was developed in collaboration with other co-workers that was compatible with the deep screening platform. The developed cell-free bead platform was based on a novel on-bead DNA assembly technique, which has not been explored in prior art. Protocols for preparing the bead repertoires were optimized and novel bead coupling chemistries were established for orthogonal tethering of DNA and protein on the beads. The developed technology was characterized in bead library selections using fluorescently labeled target molecule and flow cytometric sorting to enrich target binding molecules from the population.
The feasibility of the deep screening platform, named PIDA (protein interaction dependent assembly), was studied using SpyCatcher/SpyTag binder pair as the model system. Spycatcher and SpyTag are parts of a split bacterial adhesin. The two parts form a covalent bond with each other upon association. Major part of the work aiming towards PIDA-assay was to design DNA recovery methods for the unambiguous amplification of correctly assembled DNA avoiding unspecific background amplification. After optimization of the reaction conditions, correct pairing of SpyCatcher-DNA and SpyTag-DNA on the surface of microbeads was verified by agarose gel analysis and sequencing confirming the feasibility of the PIDA-technology. SpyCatcher-DNA paired correctly with the target peptide, i.e. SpyTag, with 75% efficiency when non-target peptides were provided in the same reaction. It was also observed that polyethylene glycol could be used to improve the interaction specificity. Due to technical challenges intrinsic to the method and the limited time frame the work was focused on optimizing the proof-of-concept experiments. Consequently, the benefits of PIDA-assay in comparison to other techniques in the art remain to be shown in large scale library studies.
In parallel to the sequencing-based PIDA-assay, an analogous microbead platform was developed, based on optical signal analysis with flow cytometry. The developed color-coded microbead screening platform was applied to screening anti-digoxigenin ScFv clone repertoires for affinity-improved clones. Ten-fold improvement in digoxigenin binding affinity was obtained by the optical bead screening platform. Manuscripts on the novel bead surface display method using on-bead DNA assembly and the color-coded suspension bead array technology have been prepared to be submitted for peer-review.
