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AssigneeThe Scripps Research Institute
InventorsCarlos F. Barbas, III, Joel M. Gottesfeld, Peter E. Wright
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Zinc finger proteins of the Cys.sub.2 His.sub.2 type represent a class of malleable DNA binding proteins which may be selected to bind diverse sequences. Typically, zinc finger proteins containing three zinc finger domains, like the murine transcription factor Zif268 and the human transcription factor Sp1, bind nine contiguous base pairs (bp). To create a class of proteins which would be generally applicable to target unique sites within complex genomes, the present invention provides a polypeptide linker that fuses two three-finger proteins. Two six-fingered proteins were created and demonstrated to bind 18 contiguous bp of DNA in a sequence specific fashion. Expression of these proteins as fusions to activation or repression domains allows transcription to be specifically up or down modulated within cells. Polydactyl zinc finger proteins are broadly applicable as genome-specific transcriptional switches in gene therapy strategies and the development of novel transgenic plants and animals. Such proteins are useful for inhibiting, activating or enhancing gene expression from a zinc finger-nucleotide binding motif containing promoter or other transcriptional control element, as well as a structural gene or RNA sequence.
An isolated zinc finger-nucleotide binding polypeptide variant comprising at least three zinc finger modules that bind to a target cellular nucleotide sequence and modulate the transcriptional function of the cellular nucleotide sequence, wherein the amino acid sequence of each zinc finger module that binds a target cellular nucleotide comprises two cysteines and two histidines whereby both cysteines are amino proximal to both histidines and wherein each of three modules of said variant has at least one amino acid sequence modification.
An isolated zinc finger-nucleotide binding polypeptide variant having at least six zinc finger modules that bind to a target cellular nucleotide sequence and modulate the transcriptional function of the cellular nucleotide sequence, wherein each zinc finger module that binds a target cellular nucleotide sequence is modified, wherein the polypeptide binds to a cellular nucleotide sequence comprising two 9-base pair binding sites and wherein the two 9-base pair binding sites are separated by a variable number of nucleotides.
A nucleotide sequence encoding a zinc finger nucleotide binding polypeptide variant having the zinc finger modules that bind to a target cellular nucleotide sequence and modulate the transcriptional function of the cellular nucleotide sequence, wherein each zinc finger module that binds a target cellular nucleotide sequence is modified comprising a transcriptional activation domain in operable linkage with the nucleotide sequence, wherein the transcriptional activational domain is a herpes simplex virus VP16 protein.
A nucleotide sequence encoding a zinc finger-nucleotide binding polypeptide variant having zinc finger modules that bind to a target cellular nucleotide sequence and modulate the transcriptional function of the cellular nucleotide sequence, wherein each zinc finger module that binds a target cellular nucleotide sequence is modified comprising a repressor domain in operable linkage with the nucleotide sequence, wherein the repressor domain is the Kruppel-associated box A domain (KRAB-A).
An in vitro method for inhibiting a transcriptional function of a target cellular nucleotide sequence comprising a zinc finger-nucleotide binding motif, the method comprising contacting the motif with an effective amount of a zinc finger-nucleotide binding polypeptide variant comprised of at least three zinc finger molecules, wherein the amino acid sequence of each zinc finger module that binds the motif comprises two cysteines and two histidines whereby both cysteines are amino proximal to both histidines and wherein each of three modules has at least one amino acid sequence modification, thereby inhibiting a transcriptional function of the sequence.
A method for isolating a zinc finger-nucleotide binding polypeptide variant which binds to a cellular nucleotide sequence comprising:
A method for identifying a zinc finger-nucleotide binding polypeptide variant comprised of at least three zinc finger modules, which modulates the transcriptional function of cellular nucleotide sequence and binds to a zinc finger-nucleotide binding motif, wherein the amino acid sequence of each module that binds to a zinc finger nucleotide binding motif comprises two cysteines and two histidines whereby both cysteines are amino proximal to both histidines and wherein each of three modules of said variant has at least one amino acid sequence modification, said method comprising:
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