The chemical transformations of biology occur in a compartmentalized context inside the cell membrane. Systematically studying this compartmentalized chemistry and harnessing its benefits for therapeutic applications through directed enzyme evolution will require methods for controlled synthesis and functional screening of cell-like compartments. Mentored research activities significantly expanded on current efforts in microfluidic directed evolution by exploring circuitry for the controlled high-throughput synthesis of monodisperse water droplets in oil for in vitro compartmentalization (IVC). This strategy Is enabling new explorations of RNA's catalytic fitness landscape by prohibiting a single advantageous genotype from dominating in the selective amplification reaction, and exaggerating neutral drift ofthe population. A nozzle array microfluidic IVC (MIVC) circuit was developed for these experiments and enabled selections encompassing l e 8 individuals per hour. Directed evolution of proteins with complex phenotypes (transport, membrane display, catalysis) will form the theme for independent phase investigations. The pIVC system will be used to synthesize monodisperse lipid vesicles for compartmentalization and functional display of integral membrane proteins, P-galactosidase and hemolysin will serve as models for using the pIVC processor to evolve new catalytic and selective transport functions on cytosolic and transmembrane proteins, respectively. Long-tennn research program goals include evolving membrane receptors (CCR5 and CD4) in lipid vesicles, selecting for enhanced binding of viral protein-receptor complexes, evolutionary structure-function studies, and synthesizing membrane-bound evolvable ligands for applications In targeted and decoy therapeutics.
Miniaturization will bring the rational control of chemistry to lipid vesicle synthesis, enabling the functional display and directed evolution of complex membrane-associated proteins such as selective transport and signaling. The vesicle arrays will be used to map mutations critical fbr viral binding to the human receptors and co-factors used by HIVrl and HCV
|Matosevic, Sandro; Paegel, Brian M (2013) Layer-by-layer cell membrane assembly. Nat Chem 5:958-63|
|Matosevic, Sandro; Paegel, Brian M (2011) Stepwise synthesis of giant unilamellar vesicles on a microfluidic assembly line. J Am Chem Soc 133:2798-800|
|Paegel, Brian M (2010) Microfluidic landscapes for evolution. Curr Opin Chem Biol 14:568-73|
|Paegel, Brian M; Joyce, Gerald F (2010) Microfluidic compartmentalized directed evolution. Chem Biol 17:717-24|