Directed Chemical Evolution of Self-Assembling Artificial Proteins Utilizing a Supramolecular System

Abstract Nature utilizes non-directed evolution to generate a vast array of biological macromolecules with remarkable diversity and functionality, though at a relatively slow pace. With advances in biotechnology, both directed and non-directed evolution approaches have been employed to design a wide range of biomacromolecules, including self-assembling artificial proteins (SAPs). However, these biological methods are limited to the natural set of twenty amino acids, resulting in SAPs that often lack functional diversity. Chemical approaches, such as micelle-assisted protein labeling technology (MAPLabTech), have enabled the creation of functional SAPs. Despite its potential, MAPLabTech remains a complex, low-throughput, and time-consuming methodology. To overcome these challenges, herein, we disclose a new method, termed S upramolecule- A ssisted P rotein Lab eling Tech nology (SAPLabTech) . In this method, γ-cyclodextrin acts as a host to solubilize hydrophobic chemical probes, forming a cyclodextrin–probe supramolecular complex that enables site-specific bioconjugation reaction and the synthesis of well-defined monodisperse SAPs in quantitative yield. The versatility of this technology is demonstrated by employing three distinct classes of chemical probes with varying warhead functionalities, linker lengths, and tail hydrophobicity to construct diverse SAP libraries with rich structural features.