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Modular binder technology by NGS-aided, high-resolution selection in yeast of designed armadillo modules
Yvonne Stark1, Faye Menard1, Jeliazko R Jeliazkov1
1Department of Biochemistry, University of Zürich, Zürich CH-8057, Switzerland.
Summary
Modular binders offer a faster, cheaper way to create diagnostic agents compared to traditional methods. Researchers developed a new yeast surface display selection strategy to generate highly specific modular binders for improved diagnostic detection.
Area of Science:
- Biotechnology
- Molecular Biology
- Protein Engineering
Background:
- Conventional binders for diagnostics are often time-consuming and costly to produce individually.
- Modular binders, designed in silico from pre-existing modules, offer a potential alternative for recognizing specific epitopes.
- Designed armadillo repeat proteins (dArmRP) serve as promising scaffolds for modular binder development.
Purpose of the Study:
- To generate highly specific modular binder modules using yeast surface display selection.
- To develop an effective selection strategy for identifying binders with high specificity, even with low abundance.
- To validate the utility of the developed modular binders as diagnostic detection agents.
Main Methods:
- A rationally designed dArmRP library was subjected to yeast surface display selection.
- A reverse-competitor strategy was employed to enhance the selection sensitivity for specificity.
- Deep sequencing and next-generation sequencing were used to analyze selection pools and identify phenotypic hits.
- A proof-of-principle study involved constructing a binder by replacing wild-type modules with newly selected ones.
Main Results:
- Highly specific dArmRP modules were successfully generated through the developed selection strategy.
- The reverse-competitor strategy effectively increased sensitivity in distinguishing specific binders from cross-reactive ones.
- Phenotypic hits with as low as 0.1% abundance were retrievable from selection pools via deep sequencing.
- A newly constructed modular binder demonstrated very high affinity for its target and was validated in western blot analysis.
Conclusions:
- The developed yeast surface display selection strategy is effective for generating highly specific modular binders.
- Modular binders, particularly dArmRPs, can be efficiently designed and validated as diagnostic detection agents.
- This approach offers a cost- and time-efficient alternative for developing novel diagnostic tools.
