Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery.


The present invention provides an in vitro system for compartmentalization of large mol. libraries and provides methods for selection and isolation of mols. having desired activities from such libraries. The present invention provides novel and inventive applications of IVC for the selection of mols. being capable of modulating a particular activity of a known biol. active moiety, including, but not limited to an enzyme. The inventors of the present invention utilize a micelle delivery system that enables the transport of various solutes, including metal ions, into the emulsion droplets thereby inducing a desired activity of the known biol. active moiety or of the gene product. The present invention is based ion part on the unexpected finding that an IVC system can be used for directed evolution of nuclease inhibitors. In vitro compartmentalization (IVC) uses water-in-oil emulsions to create artificial cell-like compartments in which genes can be individually transcribed and translated. Here, the inventors present a new application of IVC for the selection of DNA-nuclease inhibitors. They developed a nano-droplets delivery system that allows the transport of various solutes, including metal ions, into the emulsion droplets. This transport mechanism was used to regulate the activity of colicin nucleases that were co-compartmentalized with the genes, so that the nucleases were activated by nickel or cobalt ions only after the potential inhibitor genes have been translated. They demonstrated its utility by selecting libraries of the gene encoding the cognate inhibitor of colicin E9 (immunity protein 9, or Im9) for inhibition of another colicin (ColE7). The in vitro evolved inhibitors show significant inhibition of ColE7 both in vitro and in vivo. These Im9 variants carry mutations into residues that det. the selectivity of the natural counterpart (Im7) while completely retaining the residues that are conserved throughout the family of immunity protein inhibitors. [on SciFinder(R)]


CAPLUS AN 2006:469657(Patent)