Combining structure-based design with phage display to create new Cys(2)His(2) zinc finger dimers

Background: Several strategies have been reported for the design and select ion of novel DNA-binding proteins. Most of these studies have used Cys(2)Hi s(2) zinc finger proteins as a framework, and have focused on constructs th at bind DNA in a manner similar to Zif268, with neighboring fingers connect ed by a canonical (Kruppel-type) linker. This linker does not seem ideal fo r larger constructs because only modest improvements in affinity are observ ed when more than three fingers are connected in this manner. Two strategie s have been described that allow the productive assembly of more than three canonically linked fingers on a DNA site: connecting sets of fingers using linkers (covalent), or assembling sets of fingers using dimerization domai ns (non-covalent). Results: Using a combination of structure-based design and phage display, w e have developed a new dimerization system for Cys(2)His(2) zinc fingers th at allows the assembly of more than three fingers on a desired target site. Zinc finger constructs employing this new dimerization system have high af finity and good specificity for their target sites both in vitro and in viv o. Constructs that recognize an asymmetric binding site as heterodimers can be obtained through substitutions in the zinc finger and dimerization regi ons. Conclusions: Our modular zinc finger dimerization system allows more than t hree Cys(2)His(2) zinc fingers to be productively assembled on a DNA-bindin g site. Dimerization may offer certain advantages over covalent linkage for the recognition of large DNA sequences. Our results also illustrate the po wer of combining structure-based design with phage display in a strategy th at assimilates the best features of each method.