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(12) Patent Application Publication (10) Pub. No.: US 2002/0165356A1 Barbas, III Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2002/0165356A1 Barbas, III Et Al US 2002O165356A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2002/0165356A1 Barbas, III et al. (43) Pub. Date: Nov. 7, 2002 (54) ZNC FINGER BINDING DOMAINS FOR Related U.S. Application Data NUCLEOTIDE SEQUENCE ANN (63) Continuation-in-part of application No. 60/367,356, (75) Inventors: Carlos F. Barbas III, Solana Beach, filed on Feb. 21, 2001, now abandoned. CA (US); Birgit Dreier, Adlikon (CH) Publication Classification Correspondence Address: THE SCRIPPS RESEARCH INSTITUTE (51) Int. Cl." ............................ C12N 9/22; CO7H 21/04; OFFICE OF PATENT COUNSEL, TPC-8 C12P 21/02; C12N 5/06 10550 NORTH TORREY PINES ROAD (52) U.S. Cl. ........................ 530/350; 435/69.1; 435/199; LAJOLLA, CA 92037 (US) 536/23.5; 435/320.1; 435/325 (73) Assignee: The Scripps Research Institute, 10550 (57) ABSTRACT N. Torrey Pines Road, La Jolla, CA 92037 Polypeptides that contain from 2 to 12 Zinc finger-nucleotide (US) binding regions that bind to nucleotide Sequences of the formula (ANN)2-12 are provided. Polynucleotides that (21) Appl. No.: 10/080,100 encode Such polypeptides and methods of regulating gene expression with Such polypeptides and polynucleotides are (22) Filed: Feb. 21, 2002 also provided. Patent Application Publication Nov. 7, 2002. Sheet 1 of 9 US 2002/0165356 A1 Number in parenthesis = SEQID NO F N G E R 1 F N G E R 2 F N G E R 3 C7. GCG -1. 1 2 3 4 5 6 CT, GA Patent Application Publication Nov. 7, 2002 Sheet 2 of 9 US 2002/0165356A1 aara ara area as as an vers do ov Oval in r as a or er en cry S. air as as as ss N1 a1 we se 'Wen- Ne' we as o c 2 Vo as d wd a trot a lf a - un - c. s' a s rr - r2: H - as r up a CD (D - C is a F| a -(c) ra 3 -|- n - 2 a r- or Y, S-la 5- cata - - - - a - Ed H- Hist - E NC to u? to N of NC a C to ex r - g a ri N - - s - an a a ara Yn an C. red ea (r. sas or er or or er er we nea We N1 se we H road a la y y - r s 9 Y-2 ry - r A a () g enlar (Na etc.) c 3 -lone - HE- S-lin - c. r H Clar H - 4 - A a. NC Nud to af ca CN ax .2a ry ey Y Y r rar gc CD E2 l es aaaaa. as a 1-aa-ra aa a lar o H on y as rvo N oy S. N non en N. cn can on Cn in 8 wit N1 NY. N1 NY Nease N1 we sw Vo 2 te? Vd a vo - 2 - vo Y Sa E L al?o L? I a (?o a Z.es rt - s sr. A - - tr ( ) r 2, 22. r r I up to r) to d c () (DC 8 N. on n - > a to to 3 - salvag as a 3 - i. z or r 5 , o, rt A.C. - - C - - 2 eat a N COO N C CO up to cN O ?o - - N - - ve to - r cN N. N- N1/N/ n/Yen NYY1 we?wa N1 N1 p : a E- vo ( or a vo 2. P acc a? r) - a r 4, a H n rt s' -- sy a - Caz r) H a (Y) at Y up s: en E- or a 5 enlan to a 2 5 on a- gen on 3 - 2 a. <c - lar up c as a r- to- air an - to Flo |a|n z it to no oo N ea C C C CO to N ful N of ge r- - N - N - - - - us a Y - Patent Application Publication Nov. 7, 2002 Sheet 3 of 9 US 2002/0165356A1 Number in parenthesis = SEQ ID NO FIG, 3h Patent Application Publication Nov. 7, 2002 Sheet 4 of 9 US 2002/0165356A1 Number in parenthesis = SEQ ID NO AGA QLA-H-LRA AGG RSD-H-AE Patent Application Publication Nov. 7, 2002. Sheet 5 of 9 US 2002/0165356 A1 Number in parenthesis = SEQID NO FIG, 3S FIG, 3 FIG, 3W ATT TSH-G-LTT (70) ce a Cls 5 FIG, 3y Patent Application Publication Nov. 7, 2002 Sheet 7 of 9 US 2002/0165356A1 Patent Application Publication Nov. 7, 2002 Sheet 8 of 9 US 2002/0165356A1 250 2OO 150 1 OO 5 O control pcDNA Aart Aart-VP64 2C7 2C7-VP64 FIG, 5A 1 OOO 800 600 400 200 Control pcDNA Aart Aart-VP64 2C7 2C7-VP64 FIG, 5B Patent Application Publication Nov. 7, 2002. Sheet 9 of 9 US 2002/0165356A1 US 2002/0165356 A1 Nov. 7, 2002 ZNC FINGER BINDING DOMAINS FOR sequences of the 5'-ANN-3' type are found in naturally NUCLEOTIDE SEQUENCE ANN occurring proteins, like finger 5 (5'-AAA-3) of Gfi-1 Zweidler-McKay et al., (1996) Mol. Cell. Biol. 16(8), TECHNICAL FIELD OF THE INVENTION 4024-4034), finger 3 (5'-AAT-3') of YY1 Hyde-DeRuy scher, et al., (1995) Nucleic Acids Res. 23(21), 4457-4465), 0001. The field of this invention is zinc finger protein fingers 4 and 6 (5'-A/GITA-3) of CF2II Gogos et al., binding to target nucleotides. More particularly, the present (1996) PNAS 93, 2159-2164) and finger 2 (5'-AAG-3) of invention pertains to amino acid residue Sequences within TTK Fairall et al., (1993) Nature (London) 366(6454), the C-helical domain of Zinc fingers that Specifically bind to 483-7). However, in structural analysis of protein/DNA target nucleotides of the formula 5'-(ANN)-3'. complexes by X-ray or NMR studies, interaction of the amino acid residue in position 6 of the a-helix with a BACKGROUND OF THE INVENTION nucleotide other than 5" guanine was never observed. There 0002 The construction of artificial transcription factors fore, the most promising approach to identify novel Zinc has been of great interest in the past years. Gene expression finger domains binding to DNA target Sequences of the type can be specifically regulated by polydactyl Zinc finger 5'-ANN-3',5'-CNN-3" or 5'-TNN-3' is selection via phage proteins fused to regulatory domains. display. The limiting Step for this approach is the construc tion of libraries that allow the specification of a 5' adenine, 0003) Zinc finger domains of the Cys-His family have cytosine or thymine. Phage display Selections have been been most promising for the construction of artificial tran based on Zif268 in which in which different fingers of this Scription factors due to their modular structure. Each domain protein where randomized Choo et al., (1994) Proc. Natl. consists of approximately 30 amino acids and folds into a Acad. Sci. U.S.A. 91(23), 11168–72; Rebar et al., (1994) BBC. structure stabilized by hydrophobic interactions and Science (Washington, D.C., 1883-) 263(5147), 671-3; chelation of a Zinc ion by the conserved Cys-His residues. Jamieson et al., (1994) Biochemistry 33, 5689-5695; Wu et To date, the best characterized protein of this family of zinc al., (1995) PNAS 92, 344-348; Jamieson et al., (1996) Proc finger proteins is the mouse transcription factor Zif 268 Natl AcadSci USA 93, 12834-12839; Greisman et al., (1997) Pavletich et al., (1991) Science 252(5007), 809–817; Elrod Science 275(5300), 657-661). A set of 16 domains recog Erickson et al., (1996) Structure 4(10), 1171-1180). The nizing the 5'-GNN-3' type of DNA sequences has previously analysis of the Zif 268/DNA complex suggested that DNA been reported from a library where finger 2 of C7, a binding is predominantly achieved by the interaction of derivative of Zif268 U.S. Pat. No. 6,140,081, the disclosure amino acid residues of the C-helix in position -1, 3, and 6 of which is incorporated herein by reference; Wu et al., with the 3', middle, and 5' nucleotide of a 3 bp DNA subsite, (1995) PNAS 92,344-348 Wu, 1995 #164), was randomized respectively. Positions 1, 2 and 5 have been shown to make Segal et al., (1999) Proc Natl AcadSci USA 96(6), 2758 direct or water-mediated contacts with the phosphate back 2763). In Such a strategy, Selection is limited to domains bone of the DNA. Leucine is usually found in position 4 and recognizing 5'-GNN-3" or 5'-TNN-3' due to the Asp of packs into the hydrophobic core of the domain. Position 2 of finger 3 making contact with the complementary base of a 5' the C-helix has been shown to interact with other helix guanine or thymine in the finger-2. Subsite Pavletich et al., residues and, in addition, can make contact to a nucleotide (1991) Science 252(5007), 809–817; Elrod-Erickson et al., outside the 3 bp Subsite Pavletich et al., (1991) Science (1996) Structure 4(10), 1171-1180). The limited modularity 252(5007), 809–817; Elrod-Erickson et al., (1996) Structure of Zinc finger domains, which may in Some cases recognize 4(10), 1171-1180; Isalan, M. et al., (1997) Proc Natl Acad a nucleotide outside the 3 bp Subsite, has been discussed Sci USA 94(11), 5617-5621). intensively Wolfe et al., (1999) Annu. Rev. BiophyS. Biomol. 0004. The selection of modular zinc finger domains rec Struct. 3, 183-212; Segal et al., (2000) Curr Opin Chem Biol ognizing each of the 5'-GNN-3' DNA subsites with high 4(1), 34–39; Pabo et al., (2000) J.
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