Microfluidic compartmentalized directed evolution.
Directed evolution studies often make use of water-in-oil compartments, which conventionally are prepared by bulk emulsification, a crude process that generates nonuniform droplets and can damage biochemical reagents. A microfluidic emulsification circuit was devised that generates uniform water-in-oil droplets (21.9 +/- 0.8 microm radius) with high throughput (10(7)-10(8) droplets per hour). The circuit contains a radial array of aqueous flow nozzles that intersect a surrounding oil flow channel. This device was used to evolve RNA enzymes with RNA ligase activity, selecting enzymes that could resist inhibition by neomycin. Each molecule in the population had the opportunity to undergo 10(8)-fold selective amplification within its respective compartment. Then the progeny RNAs were harvested and used to seed new compartments. During five rounds of this procedure, the enzymes acquired mutations that conferred resistance to neomycin and caused some enzymes to become dependent on neomycin for optimal activity.
Paegel
Brian M
BM
Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA. briandna@scripps.edu
Joyce
Gerald F
GF
D001483:Base Sequence; D055162:Biocatalysis / Q000187:drug effects; D019020:Directed Molecular Evolution / Q000295:instrumentation*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008025:Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism; D046210:Microfluidic Analytical Techniques; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D021141:Nucleic Acid Amplification Techniques; D009821:Oils / Q000737:chemistry; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000737:chemistry
D004791:Enzyme Inhibitors; D009821:Oils; D014867:Water; D012313:RNA; D008025:Ligases; D009355:Neomycin