Work

smFASTIA: A high-sensitivity platform for purification-free kinetic screening of protein–small molecule interactions

Year: 2026

Type: article

Abstract: Elucidating the binding kinetics of protein–small molecule interactions is critical for optimizing drug efficacy and understanding mechanism of action. However, establishing structure–kinetic relationships is often hindered by the throughput of conditional assays that require protein purification. Herein, we present smFASTIA, a rapid, purification-free screening platform that integrates cell-free protein synthesis with high-sensitivity bio-layer interferometry. Using the SpyTag003–SpyCatcher003 system, we immobilized target proteins at high density directly from crude reaction mixtures, enabling the detection of weak small-molecule signals. Using human carbonic anhydrase II and its inhibitor as a model, we screened 32 variants. Instead of relying on equilibrium analysis, we utilized the initial slope of the association phase as a kinetic indicator proportional to the association rate constant. This approach successfully discriminated variants based on their kinetic profiles. Validation by surface plasmon resonance confirmed that variants classified as non-binders in our screening included those with significantly reduced on-rates, demonstrating the platform's ability to filter based on association kinetics. This workflow enables the kinetic assessment of dozens of variants within two days, providing a powerful tool for accelerating SKR studies and protein engineering. • Purification-free screening of protein-small molecule binding has been developed. • High-density protein immobilization is achieved directly from reaction mixtures. • Initial slope analysis distinguishes signals attributable to specific interactions. • The screening results showed high concordance with surface plasmon resonance data.

Source: Biochemical and Biophysical Research Communications

DOI: 10.1016/j.bbrc.2026.153300

Authors: Yuki Tokunaga, Ryo Matsunaga, Satoru Nagatoishi, Kouhei Tsumoto

Institution: The University of Tokyo

Language: English

Topic: Advanced Biosensing Techniques and Applications

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,110

Funders: Japan Agency for Medical Research and Development, Japan Society for the Promotion of Science, Japan Science and Technology Agency