Binding proteins for recognition of DNA
AssigneeGendaq Limited
InventorsYen Choo, Aaron Klug, Isidro Sánchez-Gárcia
Life of this patent
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Abstract
Disclosed are libraries of DNA sequences encoding zinc finger binding motifs for display on a particle, together with methods of designing zinc finger binding polypeptides for binding to a particular target sequence and, inter alia, use of designed zinc finger polypeptides for various in vitro or in vivo applications.
Claims49
A library of DNA sequences, each sequence encoding a zinc finger polypeptide for display, the zinc finger polypeptide comprising at least one zinc finger having partially randomised allocation of amino acids, the partially randomised zinc finger having a random allocation of amino acids at positions −1, +2, +3 and +6 and at least one of positions +1, +5 or +8, position +1 being the first amino acid in the α-helix of the zinc finger.
A library of DNA sequences, each sequence encoding a zinc finger polypeptide for display, the zinc finger polypeptide comprising at least one zinc finger having partially randomised allocation of amino acids, the partially randomised zinc finger having a random allocation of amino acids at positions −1, +1, +2, +3 and +6, position +1 being the first amino acid in the α-helix of the zinc finger.
A library of DNA sequences, each sequence encoding a zinc finger polypeptide for display, the zinc finger polypeptide comprising at least one zinc finger having partially randomised allocation of amino acids, the partially randomised zinc finger having a random allocation of amino acids at positions −1, +2, +3, +5 and +6, position +1 being the first amino acid in the α-helix of the zinc finger.
A library of DNA sequences, each sequence encoding a zinc finger polypeptide for display, the zinc finger polypeptide comprising at least one zinc finger having partially randomised allocation of amino acids, the partially randomised zinc finger having a random allocation of amino acids at positions −1, +2, +3, +6 and +8, position +1 being the first amino acid in the α-helix of the zinc finger.
A method of altering expression of a chromosomal gene in an isolated cell, the method comprising the step of:
- contacting a target DNA site in the chromosomal gene with a non-naturally occurring zinc finger protein comprising at least one designed zinc finger, thereby altering expression of the chromosomal gene, wherein the designed zinc finger is obtained by:
- screening a plurality of zinc finger polypeptides encoded by a library of polynucleotides for binding to a sequence comprising the target DNA site, wherein the zinc finger polypeptides each comprise three zinc fingers and at least the second zinc finger is partially randomized in that it comprises a random allocation of amino acids at positions −1, +2, +3 and +6 and at least one of positions +1, +5 or +8, wherein position +1 is the first amino acid of the alpha-helix of the zinc finger, and
- selecting the partially randomized zinc fingers that bind to the target DNA site.
Dependencies are inferred from the claim text.