Yuichi Onda ,  Yurika Ochi ,  Toshihiro Araki ,  Miho Kageoka ,  Shuzo Takeda ,  Kazunori Yamada ,  Takehiko Ueda ,  Ken Ohno ,  Minoru Tanaka ,  Daiki Sakai ,  Miki Hasegawa ,  Yoshihito Tanaka 

bioRxiv - Synthetic Biology

DOI: 10.1101/2025.11.26.690606

Abstract

Targeted protein degradation (TPD), including proteolysis targeting chimeras (PROTACs) and molecular glue degraders (MGDs), is a promising therapeutic approach. However, systematic discovery of such small molecules remains a major challenge. Here, we present PhenoDEL, a novel phenotypic DNA-encoded library (DEL) screening platform that integrates one-bead one-compound DEL (OBOC-DEL) with the Beacon® optofluidic system for high-throughput, single-cell analysis. By co-culturing individual OBOC-DEL beads and engineered reporter cells in nanoliter-scale chambers, PhenoDEL enables direct observation of compound-induced protein degradation at single-cell resolution. We demonstrate this approach by identifying compounds that induce degradation of FKBP12F36V-EGFP fusion proteins in PC-3 cells. The workflow allows precise linkage between compound identity and cellular phenotype via DNA barcoding and next-generation sequencing. PhenoDEL overcomes limitations of conventional screening methods, offering high sensitivity, spatial control, and scalability. This platform holds significant potential for mechanism-driven drug discovery, including identification of novel PROTACs and MGDs.

Summary

This preprint introduces PhenoDEL, a novel phenotypic screening platform that integrates One-Bead One-Compound DNA-Encoded Library (OBOC-DEL) technology with the Beacon® optofluidic system for high-throughput, single-cell analysis of targeted protein degradation (TPD). The platform enables direct observation of compound-induced protein degradation by co-culturing individual OBOC-DEL beads with engineered reporter cells in nanoliter-scale chambers (NanoPens). Upon UV-A irradiation, compounds are photoreleased from beads and diffuse to the cell, causing degradation of a FKBP12F36V-EGFP fusion protein. Beads associated with cells showing EGFP fluorescence loss are recovered, and their DNA barcodes are sequenced to identify active compounds. The authors optimized compound release kinetics (200 ms UV exposure), retention conditions (halting CO₂ flow increases concentration 5-fold), and imaging protocols. In a proof-of-concept screen, PhenoDEL successfully identified PROTAC molecules with >99% cell viability, demonstrating its capacity for mechanism-driven discovery of protein degraders including PROTACs and molecular glues.

Highlights

• Single-cell resolution screening: PhenoDEL achieves 1:1 pairing of individual OBOC-DEL beads and cells in 0.75 nL NanoPen chambers, enabling direct linkage between compound identity and cellular phenotype via DNA barcoding.
• Real-time quality control: The platform excludes dead or damaged cells by monitoring EGFP fluorescence before and after compound release, reducing false positives and improving data reliability compared to pooled screening methods.
• Optimized compound delivery: UV-A irradiation (390 nm, 200 ms) cleaves photolabile linkers to achieve biologically relevant concentrations (10-90 µM) while stopping CO₂ flow enhances compound retention within chambers.
• High throughput: Up to four OptoSelect® chips (3,500 nanopens/chip) can run simultaneously, enabling >10,000 samples per run; scalable to 80,000 compounds using 20k-nanopen chips.
• Validated proof-of-concept: Engineered PC-3 cells expressing FKBP12F36V-EGFP showed robust degradation of the fusion protein within 6 hours of PROTAC FKBP Degrader-3 exposure, with minimal cytotoxicity and stable EGFP baseline (>5,000 fluorescence units).
• Versatile applicability: The system is compatible with various cell types (suspension, adherent, primary cells, organoids) and reporter systems, positioning it for personalized medicine and comprehensive functional genomics.

Conclusion

PhenoDEL represents a significant advancement in DNA-encoded library screening by overcoming limitations of conventional affinity-based and droplet-based methods. The integration of OBOC-DEL with Beacon's optofluidic technology enables high-resolution, activity-based screening at the single-cell level, providing spatial control, real-time phenotypic tracking, and direct genotype-phenotype correlation. The platform's ability to precisely modulate compound release, maintain cell viability, and automatically filter out low-quality data points establishes a robust framework for discovering novel PROTACs, molecular glue degraders, and other proximity-inducing molecules. With demonstrated scalability and compatibility across diverse biological models, PhenoDEL holds substantial potential for next-generation drug discovery, particularly in targeting previously undruggable proteins through event-driven pharmacology.