US2003104526A1PendingUtilityA1

Position dependent recognition of GNN nucleotide triplets by zinc fingers

49
Priority: Mar 24, 1999Filed: Nov 20, 2001Published: Jun 5, 2003
Est. expiryMar 24, 2019(expired)· nominal 20-yr term from priority
Inventors:Qiang Liu
G01N 33/5758C07K 1/00C07K 14/00C07K 14/47C12N 15/102G01N 33/6875C12Q 1/6886G01N 33/5308C12N 15/62G01N 2500/04C12Q 2600/156C07K 2299/00C07K 14/4702
49
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Claims

Abstract

The specificity of binding of a zinc finger to a triplet or quadruplet nucleotide target subsite depends upon the location of the zinc finger in a multifinger protein and, hence, upon the location of its target subsite within a larger target sequence. The present disclosure provides zinc finger amino acid sequences for recognition of triplet target subsites having the nucleotide G in the 5′-most position of the subsite, that have been optimized with respect to the location of the subsite within the target site. Accordingly, the disclosure provides finger position-specific amino acid sequences for the recognition of GNN target subsites. This allows the construction of multi-finger zinc finger proteins with improved affinity and specificity for their target sequences, as well as enhanced biological activity.

Claims

exact text as granted — not AI-modified
What is claimed is:  
     
         1 . A zinc finger protein that binds to a target site, said zinc finger protein comprising a first (F1), a second (F2), and a third (F3) zinc finger, ordered F1, F2, F3 from N-terminus to C-terminus, said target site comprising, in 3′ to 5′ direction, a first (S1), a second (S2), and a third (S3) target subsite, each target subsite having the nucleotide sequence GNN, wherein if S1 comprises GAA, F1 comprises the amino acid sequence QRSNLVR; if S2 comprises GAA, F2 comprises the amino acid sequence QSGNLAR; if S3 comprises GAA, F3 comprises the amino acid sequence QSGNLAR; if S1 comprises GAG, F1 comprises the amino acid sequence RSDNLAR; if S2 comprises GAG, F2 comprises the amino acid sequence RSDNLAR; if S3 comprises GAG, F3 comprises the amino acid sequence RSDNLTR; if S1 comprises GAC, F 1  comprises the amino acid sequence DRSNLTR; if S2 comprises GAC, F2 comprises the amino acid sequence DRSNLTR; if S3 comprises GAC, F3 comprises the amino acid sequence DRSNLTR; if S1 comprises GAT, F1 comprises the amino acid sequence QSSNLAR; if S2 comprises GAT, F2 comprises the amino acid sequence TSGNLVR; if S3 comprises GAT, F3 comprises the amino acid sequence TSANLSR; if S1 comprises GGA, F1 comprises the amino acid sequence QSGHLAR; if S2 comprises GGA, F2 comprises the amino acid sequence QSGHLQR; if S3 comprises GGA, F3 comprises the amino acid sequence QSGHLQR; if S1 comprises GGG, F1 comprises the amino acid sequence RSDHLAR; if S2 comprises GGG, F2 comprises the amino acid sequence RSDHLSR; if S3 comprises GGG, F3 comprises the amino acid sequence RSDHLSR; if S1 comprises GGC, F1 comprises the amino acid sequence DRSHLRT; if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR; if S1 comprises GGT, F1 comprises the amino acid sequence QSSHLTR; if S2 comprises GGT, F2 comprises the amino acid sequence TSGHLSR; if S3 comprises GGT, F3 comprises the amino acid sequence TSGHLVR; if S1 comprises GCA, F1 comprises the amino acid sequence QSGSLTR; if S2 comprises GCA, F2 comprises QSGDLTR; if S3 comprises GCA, F3 comprises QSGDLTR; if S1 comprises GCG, F1 comprises the amino acid sequence RSDDLTR; if S2 comprises GCG, F2 comprises the amino acid sequence RSDDLQR; if S3 comprises GCG, F3 comprises the amino acid sequence RSDDLTR; if S1 comprises GCC, F1 comprises the amino acid sequence ERGTLAR; if S2 comprises GCC, F2 comprises the amino acid sequence DRSDLTR; if S3 comprises GCC, F3 comprises the amino acid sequence DRSDLTR; if S1 comprises GCT, F1 comprises the amino acid sequence QSSDLTR; if S2 comprises GCT, F2 comprises the amino acid sequence QSSDLTR; if S3 comprises GCT, F3 comprises the amino acid sequence QSSDLQR; if S1 comprises GTA, F1 comprises the amino acid sequence QSGALTR; if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR; if S1 comprises GTG, F1 comprises the amino acid sequence RSDALTR; if S2 comprises GTG, F2 comprises the amino acid sequence RSDALSR; if S3 comprises GTG, F3 comprises the amino acid sequence RSDALTR; if S1 comprises GTC, F1 comprises the amino acid sequence DRSALAR; if S2 comprises GTC, F2 comprises the amino acid sequence DRSALAR; and if S3 comprises GTC, F3 comprises the amino acid sequence DRSALAR.  
     
     
         2 . The zinc finger protein of  claim 1 , wherein S1 comprises GAA and F1 comprises the amino acid sequence QRSNLVR.  
     
     
         3 . The zinc finger protein of  claim 1 , wherein S2 comprises GAA and F2 comprises the amino acid sequence QSGNLAR.  
     
     
         4 . The zinc finger protein of  claim 1 , wherein S3 comprises GAA and F3 comprises the amino acid sequence QSGNLAR.  
     
     
         5 . The zinc finger protein of  claim 1 , wherein S1 comprises GAG and F1 comprises the amino acid sequence RSDNLAR.  
     
     
         6 . The zinc finger protein of  claim 1 , wherein S2 comprises GAG and F2 comprises the amino acid sequence RSDNLAR.  
     
     
         7 . The zinc finger protein of  claim 1 , wherein S3 comprises GAG and F3 comprises the amino acid sequence RSDNLTR.  
     
     
         8 . The zinc finger protein of  claim 1 , wherein S1 comprises GAC and F1 comprises the amino acid sequence DRSNLTR.  
     
     
         9 . The zinc finger protein of  claim 1 , wherein S2 comprises GAC and F2 comprises the amino acid sequence DRSNLTR.  
     
     
         10 . The zinc finger protein of  claim 1 , wherein S3 comprises GAC and F3 comprises the amino acid sequence DRSNLTR.  
     
     
         11 . The zinc finger protein of  claim 1 , wherein S1 comprises GAT and F1 comprises the amino acid sequence QSSNLAR.  
     
     
         12 . The zinc finger protein of  claim 1 , wherein S2 comprises GAT and F2 comprises the amino acid sequence TSGNLVR.  
     
     
         13 . The zinc finger protein of  claim 1 , wherein S3 comprises GAT and F3 comprises the amino acid sequence TSANLSR.  
     
     
         14 . The zinc finger protein of  claim 1 , wherein S1 comprises GGA and F1 comprises the amino acid sequence QSGHLAR.  
     
     
         15 . The zinc finger protein of  claim 1 , wherein S2 comprises GGA and F2 comprises the amino acid sequence QSGHLQR.  
     
     
         16 . The zinc finger protein of  claim 1 , wherein S3 comprises GGA and F3 comprises the amino acid sequence QSGHLQR.  
     
     
         17 . The zinc finger protein of  claim 1 , wherein S1 comprises GGG and F1 comprises the amino acid sequence RSDHLAR.  
     
     
         18 . The zinc finger protein of  claim 1 , wherein S2 comprises GGG and F2 comprises the amino acid sequence RSDHLSR.  
     
     
         19 . The zinc finger protein of  claim 1 , wherein S3 comprises GGG and F3 comprises the amino acid sequence RSDHLSR.  
     
     
         20 . The zinc finger protein of  claim 1 , wherein S1 comprises GGC and F1 comprises the amino acid sequence DRSHLTR.  
     
     
         21 . The zinc finger protein of  claim 1 , wherein S2 comprises GGC and F2 comprises the amino acid sequence DRSHLAR.  
     
     
         22 . The zinc finger protein of  claim 1 , wherein S11 comprises GGT and F1 comprises the amino acid sequence QSSHLTR.  
     
     
         23 . The zinc finger protein of  claim 1 , wherein S2 comprises GGT and F2 comprises the amino acid sequence TSGHLSR.  
     
     
         24 . The zinc finger protein of  claim 1 , wherein S3 comprises GGT and F3 comprises the amino acid sequence TSGHLVR.  
     
     
         25 . The zinc finger protein of  claim 1 , wherein S1 comprises GCA and F1 comprises the amino acid sequence QSGSLTR.  
     
     
         26 . The zinc finger protein of  claim 1 , wherein S2 comprises GCA and F2 comprises the amino acid sequence QSGDLTR.  
     
     
         27 . The zinc finger protein of  claim 1 , wherein S3 comprises GCA and F3 comprises the amino acid sequence QSGDLTR.  
     
     
         28 . The zinc finger protein of  claim 1 , wherein S1 comprises GCG and F1 comprises the amino acid sequence RSDDLTR.  
     
     
         29 . The zinc finger protein of  claim 1 , wherein S2 comprises GCG and F2 comprises the amino acid sequence RSDDLQR.  
     
     
         30 . The zinc finger protein of  claim 1 , wherein S3 comprises GCG and F3 comprises the amino acid sequence RSDDLTR.  
     
     
         31 . The zinc finger protein of  claim 1 , wherein S1 comprises GCC and F1 comprises the amino acid sequence ERGTLAR.  
     
     
         32 . The zinc finger protein of  claim 1 , wherein S2 comprises GCC and F2 comprises the amino acid sequence DRSDLTR.  
     
     
         33 . The zinc finger protein of  claim 1 , wherein S3 comprises GCC and F3 comprises the amino acid sequence DRSDLTR.  
     
     
         34 . The zinc finger protein of  claim 1 , wherein S1 comprises GCT and F1 comprises the amino acid sequence QSSDLTR.  
     
     
         35 . The zinc finger protein of  claim 1 , wherein S2 comprises GCT and F2 comprises the amino acid sequence QSSDLTR.  
     
     
         36 . The zinc finger protein of  claim 1 , wherein S3 comprises GCT and F3 comprises the amino acid sequence QSSDLQR.  
     
     
         37 . The zinc finger protein of  claim 1 , wherein S1 comprises GTA and F1 comprises the amino acid sequence QSGALTR.  
     
     
         38 . The zinc finger protein of  claim 1 , wherein S2 comprises GTA and F2 comprises the amino acid sequence QSGALAR.  
     
     
         39 . The zinc finger protein of  claim 1 , wherein S1 comprises GTG and F1 comprises the amino acid sequence RSDALTR.  
     
     
         40 . The zinc finger protein of  claim 1 , wherein S2 comprises GTG and F2 comprises the amino acid sequence RSDALSR.  
     
     
         41 . The zinc finger protein of  claim 1 , wherein S3 comprises GTG and F3 comprises the amino acid sequence RSDALTR.  
     
     
         42 . The zinc finger protein of  claim 1 , wherein S1 comprises GTC and F1 comprises the amino acid sequence DRSALAR.  
     
     
         43 . The zinc finger protein of  claim 1 , wherein S2 comprises GTC and F2 comprises the amino acid sequence DRSALAR.  
     
     
         44 . The zinc finger protein of  claim 1 , wherein S3 comprises GTC and F3 comprises the amino acid sequence DRSALAR.  
     
     
         45 . A polypeptide comprising a zinc finger protein according to  claim 1 .  
     
     
         46 . A polypeptide according to  claim 45 , further comprising at least one functional domain.  
     
     
         47 . A polynucleotide encoding a zinc finger protein according to  claim 1 .  
     
     
         48 . A polynucleotide encoding a polypeptide according to  claim 45 .  
     
     
         49 . A polynucleotide encoding a polypeptide according to  claim 46.

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