Context sensitive paralell optimization of zinc finger dna binding domains
OwnerJ. Joung, Carl Paboinventors
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Abstract
The present invention relates to methods of identifying multi-finger Zf polypeptides that bind to a sequence of interest. Zf polypeptides identified using the methods described herein can have affinity and specificity for their target sites that is superior to those produced by alternative methods.
Claims95
A method of selecting a zinc finger polypeptide that binds to a sequence of interest comprising at least two subsites, said method comprising the steps of:
- a) incubating position-sensitive primary libraries with target site constructs under conditions sufficient to form first binding complexes, wherein said primary libraries comprise zinc finger polypeptides having one variable finger and at least one anchor finger, and wherein the target site construct has one subsite with a sequence identical to a subsite of the sequence of interest, and one or more subsites with sequences to which the anchor finger(s) bind;
- b) isolating pools comprising nucleic acid sequences encoding polypeptides, wherein said polypeptides comprise the first binding complexes;
- c) recombining the pools to produce a secondary library;
- d) incubating the secondary library with the sequence of interest under conditions sufficient to form second binding complexes;
- and
- e) isolating nucleic acid sequences encoding zinc finger polypeptides, wherein said polypeptides comprise the second binding complexes.
A method of selecting a chimeric zinc finger polypeptide that binds to a sequence interest comprising at least two subsites, said method comprising the steps of
- a) incubating position-sensitive primary libraries with target site constructs under conditions sufficient to form first binding complexes, wherein the position-sensitive primary libraries comprise zinc finger polypeptides having one variable finger and at least one anchor finger, and wherein the target site construcs have one subsite with a sequence identical to a subsite of the sequence of interest, and one or more subsites with sequences to which the anchor finger(s) bind;
- b) recombining said pools to produce a secondary library;
- c) incubating said secondary library with the sequence of interest under conditions sufficient to form second binding complexes;
- d) isolating nucleic acid sequences encoding multi-finger zinc finger polypeptides, wherein said polypeptides comprise the second binding complexes, and
- e) fusing a nucleic acid sequence encoding a functional domain to the nucleic acid sequence encoding the multi-finger zinc finger polypeptides, to form a nucleic acid encoding a chimeric multi-finger zinc finger polypeptide
A position-sensitive primary library comprising zinc finger polypeptides having one variable finger and at least one anchor finger, wherein the position of the variable finger is the same as the position of the corresponding zinc finger in a multi-finger zinc finger polypeptide.
Dependencies are inferred from the claim text.