AU2002239295A1 - Position dependent recognition of GNN nucleotide triplets by zinc fingers - Google Patents

Position dependent recognition of GNN nucleotide triplets by zinc fingers

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AU2002239295A1
AU2002239295A1 AU2002239295A AU2002239295A AU2002239295A1 AU 2002239295 A1 AU2002239295 A1 AU 2002239295A1 AU 2002239295 A AU2002239295 A AU 2002239295A AU 2002239295 A AU2002239295 A AU 2002239295A AU 2002239295 A1 AU2002239295 A1 AU 2002239295A1
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amino acid
acid sequence
zinc finger
finger protein
rsdnlar
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Andrew Jamieson
Qiang Liu
Edward Rebar
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Sangamo Therapeutics Inc
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Sangamo Therapeutics Inc
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POSITION DEPENDENT RECOGNITION OF GNN NUCLEOTIDE TRIPLETS BY ZINC FINGERS
BACKGROUND
Zinc finger proteins (ZFPs) are proteins that can bind to DNA in a sequence-specific manner. Zinc fingers were first identified in the transcription factor TFIIIA from the oocytes of the African clawed toad, Xenopus laevis. An exemplary motif characterizing one class of these protein (C2H2 class) is -Cys- (X)2-4-Cys-(X)i2-His-(X)3-5-His (where X is any amino acid) (SEQ. ID. No: 1). A single finger domain is about 30 amino acids in length, and several structural studies have demonstrated that it contains an alpha helix containing the two invariant histidine residues and two invariant cysteine residues in a beta turn coordinated through zinc. To date, over 10,000 zinc finger sequences have been identified in several thousand known or putative transcription factors. Zinc finger domains are involved not only in DNA-recognition, but also in RNA binding and in protein-protein binding. Current estimates are that this class of molecules will constitute about 2% of all human genes.
The x-ray crystal structure of Zif268, a three-finger domain from a murine transcription factor, has been solved in complex with a cognate DNA sequence and shows that each finger can be superimposed on the next by a periodic rotation. The structure suggests that each finger interacts independently with DNA over 3 base-pair intervals, with side-chains at positions -1, 2 , 3 and 6 on each recognition helix making contacts with their respective DNA triplet subsites. The amino terminus of Zif268 is situated at the 3' end of the DNA strand with which it makes most contacts. Some zinc fingers can bind to a fourth base in a target segment. If the strand with which a zinc finger protein makes most contacts is designated the target strand, some zinc finger proteins bind to a three base triplet in the target strand and a fourth base on the nontarget strand. The fourth base is complementary to the base immediately 3 ' of the three base subsite.
The structure of the Zif268-DNA complex also suggested that the DNA sequence specificity of a zinc finger protein might be altered by making amino acid substitutions at the four helix positions (-1, 2, 3 and 6) on each of the zinc finger recognition helices. Phage display experiments using zinc finger combinatorial libraries to test this observation were published in a series of papers in 1994 (Rebar et al., Science 263, 671-673 (1994); Jamieson et al., Biochemistry 33, 5689-5695 (1994); Choo et al, PNAS 91, 11163-11167 (1994)). Combinatorial libraries were constructed with randomized side-chains in either the first or middle finger of Zif268 and then used to select for an altered Zif268 binding site in which the appropriate DNA sub-site was replaced by an altered DNA triplet. Further, correlation between the nature of introduced mutations and the resulting alteration in binding specificity gave rise to a partial set of substitution rules for design of ZFPs with altered binding specificity.
Greisman & Pabo, Science 275, 657-661 (1997) discuss an elaboration of the phage display method in which each finger of a Zif268 was successively randomized and selected for binding to a new triplet sequence. This paper reported selection of ZFPs for a nuclear hormone response element, a p53 target site and a TATA box sequence.
A number of papers have reported attempts to produce ZFPs to modulate particular target sites. For example, Choo et al., Nature 372, 645 (1994), report an attempt to design a ZFP that would repress expression of a bcr-abl oncogene. The target segment to which the ZFPs would bind was a nine base sequence 5'GCA GAA GCC3 ' chosen to overlap the junction created by a specific oncogenic translocation fusing the genes encoding bcr and abl. The intention was that a ZFP specific to this target site would bind to the oncogene without binding to abl or bcr component genes. The authors used phage display to screen a mini-library of variant ZFPs for binding to this target segment. A variant ZFP thus isolated was then reported to repress expression of a stably transfected bcr-able construct in a cell line.
Pomerantz et al., Science 261, 93-96 (1995) reported an attempt to design a novel DNA binding protein by fusing two fingers from Zif268 with a homeodomain from Oct-1. The hybrid protein was then fused with a transcriptional activator for expression as a chimeric protein. The chimeric protein was reported to bind a target site representing a hybrid of the subsites of its two components. The authors then constructed a reporter vector containing a luciferase gene operably linked to a promoter and a hybrid site for the chimeric DNA binding protein in proximity to the promoter. The authors reported that their chimeric DNA binding protein could activate expression of the luciferase gene. Liu et al., PNAS 94, 5525-5530 (1997) report forming a composite zinc finger protein by using a peptide spacer to link two component zinc finger proteins each having three fingers. The composite protein was then further linked to transcriptional activation domain. It was reported that the resulting chimeric protein bound to a target site formed from the target segments bound by the two component zinc finger proteins. It was further reported that the chimeric zinc finger protein could activate transcription of a reporter gene when its target site was inserted into a reporter plasmid in proximity to a promoter operably linked to the reporter.
Choo et al., WO 98/53058, WO98/53059, and WO 98/53060 (1998) discuss selection of zinc finger proteins to bind to a target site within the HIN Tat gene. Choo et al. also discuss selection of a zinc finger protein to bind to a target site encompassing a site of a common mutation in the oncogene ras. The target site within ras was thus constrained by the position of the mutation.
Previously-disclosed methods for the design of sequence-specific zinc finger proteins have often been based on modularity of individual zinc fingers; i.e., the ability of a zinc finger to recognize the same target subsite regardless of the location of the finger in a multi-finger protein. Although, in many instances, a zinc finger retains the same sequence specificity regardless of its location within a multi-finger protein; in certain cases, the sequence specificity of a zinc finger depends on its position. For example, it is possible for a finger to recognize a particular triplet sequence when it is present as finger 1 of a three-finger protein, but to recognize a different triplet sequence when present as finger 2 of a three- finger protein.
Attempts to address situations in which a zinc finger behaves in a non- modular fashion {i.e., its sequence specificity depends upon its location in a multi- finger protein) have, to date, involved strategies employing randomization of key binding residues in multiple adjacent zinc fingers, followed by selection. See, for example, Isalan et al. (2001) Nature Biotechnol. 19:656-660. However, methods for rational design of polypeptides containing non -modular zinc fingers have not heretofore been described. SUMMARY The present disclosure provides compositions comprising and methods involving position dependent recognition of GNN nucleotide triplets by zinc fingers. Thus, provided herein is a zinc finger protein that binds to a target site, said zinc finger protein comprising a first (FI), a second (F2), and a third (F3) zinc finger, ordered FI, F2, F3 from N-terminus to C-terminus, said target site comprising, in 3' to 5' direction, a first (SI), a second (S2), and a third (S3) target subsite, each target subsite having the nucleotide sequence GNN, wherein if S 1 comprises GAA, FI comprises the amino acid sequence QRSNLNR; if S2 comprises GAA, F2 comprises the amino acid sequence QSGΝLAR; if S3 comprises GAA, F3 comprises the amino acid sequence QSGΝLAR; if SI comprises GAG, F 1 comprises the amino acid sequence RSDΝLAR; if S2 comprises GAG, F2 comprises the amino acid sequence RSDΝLAR; if S3 comprises GAG, F3 comprises the amino acid sequence RSDΝLTR; if SI comprises GAC, FI comprises the amino acid sequence DRSΝLTR; if S2 comprises GAC, F2 comprises the amino acid sequence DRSΝLTR; if S3 comprises GAC, F3 comprises the amino acid sequence DRSΝLTR; if SI comprises GAT, FI comprises the amino acid sequence QSSΝLAR; if S2 comprises GAT, F2 comprises the amino acid sequence TSGΝLNR; if S3 comprises GAT, F3 comprises the amino acid sequence TSAΝLSR; if S 1 comprises GGA, FI 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 SI comprises GGG, FI 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 SI comprises GGC, FI comprises the amino acid sequence DRSHLRT; if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR; if SI comprises GGT, FI 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 TSGHLNR; if SI comprises GCA, FI comprises the amino acid sequence QSGSLTR; if S2 comprises GCA, F2 comprises QSGDLTR; if S3 comprises GCA, F3 comprises QSGDLTR; if S 1 comprises GCG, F 1 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 SI comprises GCC, FI 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 S 1 comprises GCT, F 1 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 SI comprises GTA, FI comprises the amino acid sequence QSGALTR; if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR; if SI comprises GTG, FI 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 SI comprises GTC, FI 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.
Also provided are methods of designing a zinc finger protein comprising a first (FI), a second (F2), and a third (F3) zinc finger, ordered FI, F2, F3 from N- terminus to C-terminus that binds to a target site comprising, in 3' to 51 direction, a first (SI), a second (S2), and a third (S3) target subsite, each target subsite having the nucleotide sequence GNN, the method comprising the steps of (a) selecting the FI zinc finger such that it binds to the SI target subsite, wherein if SI comprises GAA, FI comprises the amino acid sequence QRSNLNR; if SI comprises GAG, FI comprises the amino acid sequence RSDΝLAR; if SI comprises GAC, FI comprises the amino acid sequence DRSΝLTR; if SI comprises GAT, FI comprises the amino acid sequence QSSΝLAR; if SI comprises GGA, FI comprises the amino acid sequence QSGHLAR; if SI comprises GGG, FI comprises the amino acid sequence RSDHLAR; if SI comprises GGC, FI comprises the amino acid sequence DRSHLRT; if SI comprises GGT, FI comprises the amino acid sequence QSSHLTR; if SI comprises GCA, FI comprises QSGSLTR; if SI comprises GCG, FI comprises RSDDLTR; if S2 comprises GCG, F2 comprises RSDDLQR; if SI comprises GCC, FI comprises ERGTLAR; if SI comprises GCT, FI comprises the amino acid sequence QSSDLTR; if SI comprises GTA, FI comprises the amino acid sequence QSGALTR; if SI comprises GTG, FI comprises the amino acid sequence RSDALTR; if SI comprises GTC, FI comprises the amino acid sequence DRSALAR; (b) selecting the F2 zinc finger such that it binds to the S2 target subsite, wherein S2 comprises GAA, F2 comprises the amino acid sequence QSGNLAR; if S2 comprises GAG, F2 comprises the amino acid sequence RSDNLAR; if S2 comprises GAC, F2 comprises the amino acid sequence DRSNLTR; if S2 comprises GAT, F2 comprises the amino acid sequence TSGNLNR; if S2 comprises GGA, F2 comprises the amino acid sequence QSGHLQR; if S2 comprises GGG, F2 comprises the amino acid sequence RSDHLSR; if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR; if S2 comprises GGT, F2 comprises the amino acid sequence TSGHLSR; if S2 comprises GCA, F2 comprises the amino acid sequence QSGDLTR; if S2 comprises GCC, F2 comprises the amino acid sequence DRSDLTR; if S2 comprises GCT, F2 comprises the amino acid sequence QSSDLTR; if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR; if S2 comprises GTG, F2 comprises the amino acid sequence RSDALSR; if S2 comprises GTC, F2 comprises the amino acid sequence DRSALAR; and (c) selecting the F3 zinc finger such that it binds to the S3 target subsite, wherein if S3 comprises GAA, F3 comprises the amino acid sequence QSGΝLAR; if S3 comprises GAG, F3 comprises the amino acid sequence RSDΝLTR; if S3 comprises GAC, F3 comprises the amino acid sequence DRSΝLTR; if S3 comprises GAT, F3 comprises the amino acid sequence TSAΝLSR; if S3 comprises GGA, F3 comprises the amino acid sequence
QSGHLQR; if S3 comprises GGG, F3 comprises RSDHLSR; if S3 comprises GGT, F3 comprises the amino acid sequence TSGHLNR; if S3 comprises GCA, F3 comprises the amino acid sequence QSGDLTR; if S3 comprises GCG, F3 comprises the amino acid sequence RSDDLTR; if S3 comprises GCC, F3 comprises the amino acid sequence DRSDLTR; if S3 comprises GCT, F3 comprises the amino acid sequence QSSDLQR; if S3 comprises GTG, F3 comprises RSDALTR; and if S3 comprises GTC, F3 comprises the amino acid sequence DRSALAR; thereby designing a zinc finger protein that binds to a target site. In certain embodiments of the zinc finger proteins and methods described herein, SI comprises GAA and FI comprises the amino acid sequence QRSNLVR. In other embodiments, S2 comprises GAA and F2 comprises the amino acid sequence QSGNLAR. In other embodiments, S3 comprises GAA and F3 comprises the amino acid sequence QSGNLAR. In other embodiments, SI comprises GAG and FI comprises the amino acid sequence RSDNLAR. In other embodiments, S2 comprises GAG and F2 comprises the amino acid sequence RSDNLAR. In other embodiments, S3 comprises GAG and F3 comprises the amino acid sequence RSDNLTR. In other embodiments, S 1 comprises GAC and FI comprises the amino acid sequence DRSNLTR. In other embodiments, S2 comprises GAC and F2 comprises the amino acid sequence DRSNLTR. In other embodiments, S3 comprises GAC and F3 comprises the amino acid sequence DRSNLTR. In other embodiments, SI comprises GAT and FI comprises the amino acid sequence QSSNLAR. In other embodiments, S2 comprises GAT and F2 comprises the amino acid sequence TSGNLNR. In other embodiments, S3 comprises GAT and F3 comprises the amino acid sequence TSAΝLSR. In other embodiments, SI comprises GGA and FI comprises the amino acid sequence QSGHLAR. In other embodiments, S2 comprises GGA and F2 comprises the amino acid sequence QSGHLQR. In other embodiments, S3 comprises GGA and F3 comprises the amino acid sequence QSGHLQR. In other embodiments, SI comprises GGG and FI comprises the amino acid sequence RSDHLAR. In other embodiments, S2 comprises GGG and F2 comprises the amino acid sequence RSDHLSR. In other embodiments, S3 comprises GGG and F3 comprises the amino acid sequence RSDHLSR. In other embodiments, SI comprises GGC and FI comprises the amino acid sequence DRSHLTR. In other embodiments, S2 comprises GGC and F2 comprises the amino acid sequence DRSHLAR. In other embodiments, SI comprises GGT and FI comprises the amino acid sequence QSSHLTR. In other embodiments, S2 comprises GGT and F2 comprises the amino acid sequence TSGHLSR. In other embodiments, S3 comprises GGT and F3 comprises the amino acid sequence TSGHLVR. In other embodiments, SI comprises GCA and FI comprises the amino acid sequence QSGSLTR. In other embodiments, S2 comprises GCA and F2 comprises the amino acid sequence QSGDLTR. In other embodiments, S3 comprises GCA and F3 comprises the amino acid sequence QSGDLTR. In other embodiments, SI comprises GCG and FI comprises the amino acid sequence RSDDLTR. In other embodiments, S2 comprises GCG and F2 comprises the amino acid sequence RSDDLQR. In other embodiments, S3 comprises GCG and F3 comprises the amino acid sequence RSDDLTR. In other embodiments, SI comprises GCC and FI comprises the amino acid sequence ERGTLAR. In other embodiments, S2 comprises GCC and F2 comprises the amino acid sequence DRSDLTR. In other embodiments, S3 comprises GCC and F3 comprises the amino acid sequence DRSDLTR. In other embodiments, SI comprises GCT and FI comprises the amino acid sequence QSSDLTR. In other embodiments, S2 comprises GCT and F2 comprises the amino acid sequence QSSDLTR. In other embodiments, S3 comprises GCT and F3 comprises the amino acid sequence QSSDLQR. In other embodiments, SI comprises GTA and FI comprises the amino acid sequence QSGALTR. In other embodiments, S2 comprises GTA and F2 comprises the amino acid sequence QSGALAR. In other embodiments, SI comprises GTG and FI comprises the amino acid sequence RSDALTR. In other embodiments, S2 comprises GTG and F2 comprises the amino acid sequence RSDALSR. In other embodiments, S3 comprises GTG and F3 comprises the amino acid sequence RSDALTR. In other embodiments, SI comprises GTC and FI comprises the amino acid sequence DRSALAR. In other embodiments, S2 comprises GTC and F2 comprises the amino acid sequence DRSALAR. In other embodiments, S3 comprises GTC and F3 comprises the amino acid sequence DRSALAR.
Also provided are polypeptides comprising any of zinc finger proteins described herein. In certain embodiments, the polypeptide further comprises at least one functional domain. Also provided are polynucleotides encoding any of the polypeptides described herein. Thus, also provided are nucleic acid encoding zinc fingers, including all of the zinc fingers described above.
Also provided are segments of a zinc finger comprising a sequence of seven contiguous amino acids as shown herein. Also provided are nucleic acids encoding any of these segments and zinc fingers comprising the same. Also provided are zinc finger proteins comprising first, second and third zinc fingers. The first, second and third zinc fingers comprise respectively first, second and third segments of seven contiguous amino acids as shown herein. Also provided are nucleic acids encoding such zinc finger proteins. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows results of site selection analysis of two representative zinc finger proteins (leftmost 4 columns) and measurements of binding affinity for each of these proteins to their intended target sequences and to variant target sequences, (rightmost 3 columns). Analysis of ZFP1 is shown in the upper portion of the figure and analysis of ZFP2 is shown in the lower portion of the figure. For the site selection analyses, the amino acid sequences of residues -1 through +6 of the recognition helix of each of the three component zinc fingers (F3, F2 and FI) are shown across the top row; the intended target sequence (divided into finger-specific target subsites) is shown across the second row, and a summary of the sequences bound is shown in the third row. Data for F3 is shown in the second column, data for F2 is shown in the third column, and data for FI is shown in the third column.
For the binding affinity analyses, the designed target sequence for each ZFP ("cognate") and two related sequences ("Mt") are shown (column 6), along with the Kd for binding of the ZFP to each of these sequences (column 7).
Figure 2 shows amino acid sequences of zinc finger recognition regions (amino acids -1 through +6 of the recognition helix) that bind to each of the 16 GNN triplet subsites. Three amino acid sequences are shown for each trinucleotide subsite; these correspond to optimal amino acid sequences for recognition of the subsite from each of the three positions (finger 1, FI; finger 2, F2; or finger 3, F3) in a three-finger zinc finger protein. Amino acid sequences are from N-terminal to C-terminal; nucleotide sequences are from 5' to 3'.
Also shown are site selection results for each of the 48 position-dependent GNN-recognizing zinc fingers. These show the number of times a particular nucleotide was present, at a given position, in a collection of oligonucleotide sequences bound by the finger. For example, out of 15 oligonucleotides bound by a zinc finger protein with the amino acid sequence QSGHLAR present at the finger 1 (FI) position, 15 contained a G in the 5' -most position of the subsite, 15 contained a G in the middle position of the subsite, while, at the 3 '-most position of the subsite, 10 contained an A, 3 contained a G and 2 contained a T. Accordingly, this particular amino acid sequence is optimal for binding a GGA triplet from the FI position. Figures 3A, 3B and 3C show site selection data indicating positional dependence of GCA-, GAT- and GGT-bindmg zinc fingers. The first and fourth (where applicable) rows of each figure show portions of the amino acid sequence of a designed zmc finger protein. Amino acid resιdues-1 through +6 of each α- helix are listed from left to right. The second and fifth (where applicable) rows show the target sequence, divided into three triplet subsites, one for each finger of the protein shown in the first and fourth (where applicable) rows, respectively. The third and sixth (where applicable) rows show the distribution of nucleotides m the oligonucleotides obtained by site selection with the protems shown in the first and fourth (where applicable) rows, respectively. Figure 3 A shows data for fingers designed to bind GCA; Figure 3B shows data for fingers designed to bind GAT; Figure 3C shows data for fingers designed to bind GGT.
Figures 4A and 4B show properties of the engineered ZFP EP2C. Figure 4A shows site selection data. The first row provides the ammo acid sequences of residues -1 through +6 of the recognition helices for each of the three zmc fingers of the EP2C protein. The second row shows the target sequence (5' to 3'); with the distribution of nucleotides in the oligonucleotides obtained by site selection indicated below the target sequence
Figure 4B shows in vitro and in vivo assays for the binding specificity of EP2C. The first three columns show in vitro measurements of binding affinity of EP2C to its intended target sequence and several related sequences. The first column gives the name of each sequence (2C0 is the intended target sequence, compare to Figure 4A). The second column shows the nucleotide sequence of various target sequences, with differences from the intended target sequence (2C0) highlighted. The third column shows the Kd (in nM) for binding of EP2C to each of the target sequences. K S were determined by gel shift assays, using 2- fold dilution series of EP2C. The right side of the figure (fourth column and bar graph) shows relative luciferase activities (normalized to β-galactosidase levels) in stable cell lines in which expression of EP2C is inducible. Cells were co- transfected with a vector containing a luciferase coding region under the transcriptional control of the target sequence shown in the same row of the figure, and a control vector encoding β-galactosidase Luciferase and β-galactosidase levels were measured after induction of EP2C expression. Triplicate samples were assayed and the standard deviations are shown in the bar graph. pGL3 is a luciferase-encoding vector lacking EP2C target sequences. 3B is another negative control, in which luciferase expression is under transcriptional control of sequences (3B) unrelated to the EP2C target sequence.
DEFINITIONS
A zinc finger DNA binding protein is a protein or segment within a larger protein that binds DNA in a sequence-specific manner as a result of stabilization of protein structure through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Zinc finger proteins can be engineered to recognize a selected target sequence in a nucleic acid. Any method known in the art or disclosed herein can be used to construct an engineered zinc finger protein or a nucleic acid encoding an engineered zinc finger protein. These include, but are not limited to, rational design, selection methods {e.g., phage display) random mutagenesis, combinatorial libraries, computer design, affinity selection, use of databases matching zinc finger amino acid sequences with target subsite nucleotide sequences, cloning from cDNA and/or genomic libraries, and synthetic constructions. An engineered zinc finger protein can comprise a new combination of naturally-occurring zinc finger sequences. Methods for engineering zinc finger proteins are disclosed in co-owned WO 00/41566 and WO 00/42219; as well as in WO 98/53057; WO 98/53058; WO 98/53059 and WO 98/53060; the disclosures of which are hereby incoφorated by reference in their entireties. Methods for identifying preferred target sequences, and for engineering zinc finger proteins to bind to such preferred target sequences, are disclosed in co-owned WO 00/42219. A designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. A selected zinc finger protein is a protein not found in nature whose production results primarily from an empirical process such as phage display. The term naturally-occurring is used to describe an object that can be found in nature as distinct from being artificially produced by man. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. Generally, the term naturally-occurring refers to an object as present in a non-pathological (undiseased) individual, such as would be typical for the species. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by up to several kilobases or more and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
A specific binding affinity between, for example, a ZFP and a specific target site means a binding affinity of at least 1 x IO6 M"1.
The terms "modulating expression" "inhibiting expression" and "activating expression" of a gene refer to the ability of a zinc finger protein to activate or inhibit transcription of a gene. Activation includes prevention of subsequent transcriptional inhibition (i.e., prevention of repression of gene expression) and inhibition includes prevention of subsequent transcriptional activation (i.e., prevention of gene activation). Modulation can be assayed by determining any parameter that is indirectly or directly affected by the expression of the target gene. Such parameters include, e.g., changes in RNA or protein levels, changes in protein activity, changes in product levels, changes in downstream gene expression, changes in reporter gene transcription (luciferase, CAT, beta- galactosidase, GFP (see, e.g., Mistili & Spector, Nature Biotechnology 15:961- 964 (1997)); changes in signal transduction, phosphorylation and dephosphorylation, receptor-ligand interactions, second messenger concentrations (e.g., cGMP, cAMP, IP3, and Ca2+), cell growth, neovascularization, in vitro, in vivo, and ex vivo. Such functional effects can be measured by any means known to those skilled in the art, e.g., measurement of RNA or protein levels, measurement of RNA stability, identification of downstream or reporter gene expression, e.g., via chemiluminescence, fluorescence, color-metric reactions, antibody binding, inducible markers, ligand binding assays; changes in intracellular second messengers such as cGMP and inositol triphosphate (IP3); changes in intracellular calcium levels; cytokine release, and the like.
A "regulatory domain" refers to a protein or a protein subsequence that has transcriptional modulation activity. Typically, a regulatory domain is covalently or non-covalently linked to a ZFP to modulate transcription. Alternatively, a ZFP can act alone, without a regulatory domain, or with multiple regulatory domains to modulate transcription. •
A D-able subsite within a target site has the motif 5 'NNGK3 '. A target site containing one or more such motifs is sometimes described as a D-able target site. A zinc finger appropriately designed to bind to a D-able subsite is sometimes referred to as a D-able finger. Likewise a zinc finger protein containing at least one finger designed or selected to bind to a target site including at least one D- able subsite is sometimes referred to as a D-able zinc finger protein.
DETAILED DESCRIPTION
I. General
Tables 1-5 list a collection of nonnaturally occurring zinc finger protein sequences and their corresponding target sites. The first column of each table is an internal reference number. The second column lists a 9 or 10 base target site bound by a three-finger zinc finger protein, with the target sites listed in 5 ' to 3 ' orientation. The third column provides SEQ ID NOs for the target site sequences listed in column 2. The fourth, sixth and eighth columns list amino acid residues from the first, second and third fingers, respectively, of a zinc finger protein which recognizes the target sequence listed in the second column. For each finger, seven amino acids, occupying positions -1 to +6 of the finger, are listed. The numbering convention for zinc fingers is defined below. Columns 5, 7 and 9 provide SEQ ID NOs for the amino acid sequences listed in columns 4, 6 and 8, respectively. The final column of each table lists the binding affinity {i.e., the Kd in nM) of the zinc finger protein for its target site. Binding affinities are measured as described below.
Each finger binds to a triplet of bases within a corresponding target sequence. The first finger binds to the first triplet starting from the 3' end of a target site, the second finger binds to the second triplet, and the third finger binds the third {i.e., the 5'-most) triplet of the target sequence. For example, the RSDSLTS finger (SEQ ID NO: 646) of SBS# 201 (Table 2) binds to 5'TTG3', the ERSTLTR finger (SEQ ID NO: 851) binds to5'GCC3' and the QRADLRR finger (SEQ ID NO: 1056) binds to 5'GCA3\
Table 6 lists a collection of consensus sequences for zinc fingers and the target sites bound by such sequences. Conventional one letter amino acid codes are used to designate amino acids occupying consensus positions. The symbol "X" designates a nonconsensus position that can in principle be occupied by any amino acid. In most zinc fingers of the C2H2 type, binding specificity is principally conferred by residues -1, +2, +3 and +6. Accordingly, consensus sequence determining binding specificity typically include at least these residues. Consensus sequences are useful for designing zinc fingers to bind to a given target sequence. Residues occupying other positions can be selected based on sequences in Tables 1-5, or other known zinc finger sequences. Alternatively, these positions can be randomized with a plurality of candidate amino acids and screened against one or more target sequences to refine binding specificity or improve binding specificity. In general, the same consensus sequence can be used for design of a zinc finger regardless of the relative position of that finger in a multi-finger zinc finger protein. For example, the sequence RXDNXXR can be used to design a N-terminal, central or C-terminal finger of three finger protein. However, some consensus sequences are most suitable for designing a zinc finger to occupy a particular position in a multi-finger protein. For example, the consensus sequence RXDHXXQ is most suitable for designing a C-terminal finger of a three-finger protein.
II. Characteristics of Zinc Finger Proteins
Zinc finger proteins are formed from zinc finger components. For example, zinc finger proteins can have one to thirty-seven fingers, commonly having 2, 3, 4, 5 or 6 fingers. A zinc finger protein recognizes and binds to a target site (sometimes referred to as a target segment) that represents a relatively small subsequence within a target gene. Each component finger of a zinc finger protein can bind to a subsite within the target site. The subsite includes a triplet of three contiguous bases all on the same strand (sometimes referred to as the target strand). The subsite may or may not also include a fourth base on the opposite strand that is the complement of the base immediately 3' of the three contiguous bases on the target strand. In many zinc finger proteins, a zinc finger binds to its triplet subsite substantially independently of other fingers in the same zinc finger protein. Accordingly, the binding specificity'of zinc finger protein containing multiple fingers is usually approximately the aggregate of the specificities of its component fingers. For example, if a zinc finger protein is formed from first, second and third fingers that individually bind to triplets XXX, YYY, and ZZZ, the binding specificity of the zinc finger protein is 3 'XXX YYY ZZZ5'.
The relative order of fingers in a zinc finger protein from N-terminal to C- terminal determines the relative order of triplets in the 3 ' to 5 ' direction in the target. For example, if a zinc finger protein comprises from N-terminal to C- terminal first, second and third fingers that individualy bind, respectively, to triplets 5' GAC3', 5'GTA3' and 5"GGC3' then the zinc finger protein binds to the target segment 3 'CAGATGCGG5 '. If the zinc finger protein comprises the fingers in another order, for example, second "finger, first finger, third finger, then the zinc finger protein binds to a target segment comprising a different permutation of triplets, in this example, 3ΑTGCAGCGG5' (see Berg & Shi, Science 271, 1081-1086 (1996)). The assessment of binding properties of a zinc finger protein as the aggregate of its component fingers may, in some cases, be influenced by context-dependent interactions of multiple fingers binding in the same protein.
Two or more zinc finger proteins can be linked to have a target specificity that is the aggregate of that of the component zinc finger proteins (see e.g., Kim & Pabo, PNAS 95, 2812-2817 (1998)). For example, a first zinc finger protein having first, second and third component fingers that respectively bind to XXX, YYY and ZZZ can be linked to a second zinc finger protein having first, second and third component fingers with binding specificities, AAA, BBB and CCC. The binding specificity of the combined first and second proteins is thus
3'XXXYYYZZZ AAABBBCCC5', where the underline indicates a short intervening region (typically 0-5 bases of any type). In this situation, the target site can be viewed as comprising two target segments separated by an intervening segment.
Linkage can be accomplished using any of the following peptide linkers. T G E K P: (SEQ. ID. No:2) (Liu et al., 1997, supra.); (G4S)n (SEQ. ID. No:3) (Kim et al., PNAS 93, 1156-1160 (1996.); GGRRGGGS; (SEQ. ID. No:4) LRQRDGERP; (SEQ. ID. No:5) LRQKDGGGSERP; (SEQ. ID. No:6) LRQKD(G3S)2 ERP (SEQ. ID. No:7) Alternatively, flexible linkers can be rationally designed using computer programs capable of modeling both DNA- binding sites and the peptides themselves or by phage display methods . In a further variation, noncovalent linkage can be achieved by fusing two zinc finger proteins with domains promoting heterodimer formation of the two zinc finger proteins. For example, one zinc finger protein can be fused with fos and the other with jun (see Barbas et al., WO 95/119431).
Linkage of two zinc finger proteins is advantageous for conferring a unique binding specificity within a mammalian genome. A typical mammalian diploid genome consists of 3 x 10 bp. Assuming that the four nucleotides A, C, G, and T are randomly distributed, a given 9 bp sequence is present ~23,000 times. Thus a ZFP recognizing a 9 bp target with absolute specificity would have the potential to bind to -23,000 sites within the genome. An 18 bp sequence is present once in 3.4 x 101 bp, or about once in a random DNA sequence whose complexity is ten times that of a mammalian genome.
A component finger of zinc finger protein typically contains about 30 amino acids and has the following motif (N-C) :
(SEQ. ID. No:8) Cys - (X) 2_4 -Cys -X . X . X . X . X . X . X . X . X . X . X . X-His - (X. 3-5 -
His
- 1 1 2 3 4 5 6 7 The two invariant histidine residues and two invariant cysteine residues in a single beta turn are co-ordinated through zinc (see, e.g., Berg & Shi, Science 271, 1081-1085 (1996)). The above motif shows a numbering convention that is standard in the field for the region of a zinc finger conferring binding specificity. The amino acid on the left (N-terminal side) of the first invariant His residues is assigned the number +6, and other amino acids further to the left are assigned successively decreasing numbers. The alpha helix begins at residue 1 and extends to the residue following the second conserved histidine. The entire helix is therefore of variable length, between 11 and 13 residues.
The process of designing or selecting a nonnaturally occurring or variant ZFP typically starts with a natural ZFP as a source of framework residues. The process of design or selection serves to define nonconserved positions (i.e., positions -1 to +6) so as to confer a desired binding specificity. One suitable ZFP is the DNA binding domain of the mouse transcription factor Zif268. The DNA binding domain of this protein has the amino acid sequence: YACPVESCDRRFSRSDELTRHIRIHTGQKP (FI) (SEQ. ID No:9) FQCRICMRNFSRSDHLTTHIRTHTGEKP (F2) (SEQ. ID. No: 10) FACDICGRKFARSDERKRHTKIHLRQK (F3) SEQ. ID. No:l 1) and binds to a target 5' GCG TGG GCG 3' (SEQ ID No:12).
Another suitable natural zinc finger protein as a source of framework residues is Sp-1. The Sp-1 sequence used for construction of zinc finger proteins corresponds to amino acids 531 to 624 in the Sp-1 transcription factor. This sequence is 94 amino acids in length. The amino acid sequence of Sp-1 is as follows:
PGKKKQHICHIQGCGKNYGKTSHLRAHLRWHTGERP FMCTWSYCGKRFTRSDELQRHKRTHTGEKK FACPECPKRFMRSDHLSKHIKTHQΝKKG (SEQ. ID. No: 13) Sp-1 binds to a target site 5'GGG GCG GGG3' (SEQ ID No: 14).
An alternate form of Sp-1, an Sp-1 consensus sequence, has the following amino acid sequence: meklrngsgd PGKKKQHACPECGKSFSKS SHLRAHQRTHTGERP YKCPECGKSFSRSDELQRHQRTHTGEKP
YKCPECGKSFSRSDHLSKHQRTHQNKKG (SEQ. ID. No: 15) (lower case letters are a leader sequence from Shi & Berg, Chemistry and Biology 1, 83-89. (1995). The optimal binding sequence for the Sp-1 consensus sequence is 5'GGGGCGGGG3' (SEQ ID No: 16) . Other suitable ZFPs are described below. There are a number of substitution rules that assist rational design of some zinc finger proteins (see Desjarlais & Berg, PNAS 90, 2256-2260 (1993); Choo & Klug, PNAS 91, 1 1163-11167 (1994); Desjarlais & Berg, PNAS 89, 7345-7349 (1992); Jamieson et al., supra; Choo et al., WO 98/53057, WO 98/53058; WO 98/53059; WO 98/53060). Many of these rules are supported by site-directed mutagenesis of the three-finger domain of the ubiquitous transcription factor, Sp-1 (Desjarlais and Berg, 1992; 1993). One of these rules is that a 5' G in a DNA triplet can be bound by a zinc finger incorporating arginine at position 6 of the recognition helix. Another substitution rule is that a G in the middle of a subsite can be recognized by including a histidine residue at position 3 of a zinc finger. A further substitution rule is that asparagine can be incorporated to recognize A in the middle of triplet, aspartic acid, glutamic acid, serine or threonine can be incorporated to recognize C in the middle of triplet, and amino acids with small side chains such as alanine can be incoφorated to recognize T in the middle of triplet A further substitution rule is that the 3' base of triplet subsite can be recognized by incoφorating the following amino acids at position -1 of the recognition helix: arginine to recognize G, glutamine to recognize A, glutamic acid (or aspartic acid) to recognize C, and threonine to recognize T. Although these substitution rules are useful in designing zinc finger proteins they do not take into account all possible target sites. Furthermore, the assumption underlying the rules, namely that a particular amino acid in a zinc finger is responsible for binding to a particular base in a subsite is only approximate. Context-dependent interactions between proximate amino acids in a finger or binding of multiple amino acids to a single base or vice versa can cause variation of the binding specificities predicted by the existing substitution rules.
The technique of phage display provides a largely empirical means of generating zinc finger proteins with a desired target specificity (see e.g., Rebar, US 5,789,538; Choo et al., WO 96/06166; Barbas et al., WO 95/19431 and WO 98/543111; Jamieson et al., supra). The method can be used in conjunction with, or as an alternative to rational design. The method involves the generation of diverse libraries of mutagenized zinc finger proteins, followed by the isolation of proteins with desired DNA-binding properties using affinity selection methods. To use this method, the experimenter typically proceeds as follows. First, a gene for a zinc finger protein is mutagenized to introduce diversity into regions important for binding specificity and/or affinity. In a typical application, this is accomplished via randomization of a single finger at positions -1, +2, +3, and +6, and sometimes accessory positions such as +1, +5, +8 and +10. Next, the mutagenized gene is cloned into a phage or phagemid vector as a fusion with gene III of a filamentous phage, which encodes the coat protein pill. The zinc finger gene is inserted between segments of gene III encoding the membrane export signal peptide and the remainder of pill, so that the zinc finger protein is expressed as an amino-terminal fusion with pill or in the mature, processed protein. When using phagemid vectors, the mutagenized zinc finger gene may also be fused to a truncated version of gene III encoding, minimally, the C- terminal region required for assembly of pill into the phage particle. The resultant vector library is transformed into E. coli and used to produce filamentous phage which express variant zinc finger proteins on their surface as fusions with the coat protein pill. If a phagemid vector is used, then the this step requires superinfection with helper phage. The phage library is then incubated with target DNA site, and affinity selection methods are used to isolate phage which bind target with high affinity from bulk phage. Typically, the DNA target is immobilized on a solid support, which is then washed under conditions sufficient to remove all but the tightest binding phage. After washing, any phage remaining on the support are recovered via elution under conditions which disrupt zinc finger - DNA binding. Recovered phage are used to infect fresh E. coli., which is then amplified and used to produce a new batch of phage particles. Selection and amplification are then repeated as many times as is necessary to enrich the phage pool for tight binders such that these may be identified using sequencing and/or screening methods. Although the method is illustrated for pill fusions, analogous principles can be used to screen ZFP variants as pNIII fusions.
In certain embodiments, the sequence bound by a particular zinc finger protein is determined by conducting binding reactions (see, e.g., conditions for determination of Kd, infra) between the protein and a pool of randomized double- stranded oligonucleotide sequences. The binding reaction is analyzed by an electrophoretic mobility shift assay (EMSA), in which protein-DΝA complexes undergo retarded migration in a gel and can be separated from unbound nucleic acid. Oligonucleotides which have bound the finger are purified from the gel and amplified, for example, by a polymerase chain reaction. The selection {i.e. binding reaction and EMSA analysis) is then repeated as many times as desired, with the selected oligonucleotide sequences. In this way, the binding specificity of a zinc finger protein having a particular amino acid sequence is determined. Zinc finger proteins are often expressed with a heterologous domain as fusion proteins. Common domains for addition to the ZFP include, e.g., transcription factor domains (activators, repressors, co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DΝA repair enzymes and their associated factors and modifiers; DΝA rearrangement enzymes and their associated factors and modifiers; chromatin associated proteins and their modifiers (e.g. kinases, acetylases and deacetylases); and DNA modifying enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. A preferred domain for fusing with a ZFP when the ZFP is to be used for represssing expression of a target gene is a KRAB repression domain from the human KOX-1 protein (Thiesen et al., New Biologist 2, 363-374 (1990); Margolin et al., Proc. Natl. Acad. Sci. USA 91, 4509-4513 (1994); Pengue et al., Nucl. Acids Res. 22:2908-2914 (1994); Witzgall et al., Proc. Natl. Acad. Sci. USA 91, 4514-4518 (1994). Preferred domains for achieving activation include the HSN NP16 activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962 (1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factor kappa B (Bitko & Barik, J Virol. 72:5610-5618 (1998)and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28 (1998)), or artificial chimeric functional domains such as VP64 (Seifpal et al., EMBO J. 11, 4961-4968 (1992)).
An important factor in the administration of polypeptide compounds, such as the ZFPs, is ensuring that the polypeptide has the ability to traverse the plasma membrane of a cell, or the membrane of an intra-cellular compartment such as the nucleus. Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents. However, proteins and other compounds such as liposomes have been described, which have the ability to translocate polypeptides such as ZFPs across a cell membrane.
For example, "membrane translocation polypeptides" have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane- translocating carriers. In one embodiment, homeodomain proteins have the ability to translocate across cell membranes. The shortest internalizable peptide of a homeodomain protein, Antennapedia, was found to be the third helix of the protein, from amino acid position 43 to 58 {see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996)). Another subsequence, the h (hydrophobic) domain of signal peptides, was found to have similar cell membrane translocation characteristics {see, e.g., Lin et al., J. Biol. Chem. 270:1 4255-14258 (1995)). Examples of peptide sequences which can be linked to a ZFP, for facilitating uptake of ZFP into cells, include, but are not limited to: an 11 amino acid peptide of the tat protein of HIN; a 20 residue peptide sequence which corresponds to amino acids 84-103 of the pi 6 protein {see Fahraeus et al, Current Biology 6:84 (1996)); the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et al, J. Biol. Chem. 269:10444 (1994)); the h region of a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et al, supra); or the NP22 translocation domain from HSN (Elliot & O'Hare, Cell 88:223-233 (1997)). Other suitable chemical moieties that provide enhanced cellular uptake may also be chemically linked to ZFPs.
Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules are composed of at least two parts (called "binary toxins"): a translocation or binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell. Several bacterial toxins, including Clostridium perfringens iota toxin, diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracis toxin, and pertussis adenylate cyclase (CYA), have been used in attempts to deliver peptides to the cell cytosol as internal or amino -terminal fusions (Arora et al, J. Biol. Chem., 268:3334-3341 (1993); Perelle et al, Infect. Immun., 61 :5147- 5156 (1993); Stenmark et _/., J Cell Biol. 113:1025-1032 (1991); Donnelly et al, PNAS 90:3530-3534 (1993); Carbonetti et al, Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295 (1995); Sebo et al, Infect. Immun. 63:3851-3857 (1995); Klimpel et al, PNAS U.S.A. 89:10277-10281 (1992); and Novak et al, J. Biol. Chem. 267:17186-17193 1992)).
Such subsequences can be used to translocate ZFPs across a cell membrane. ZFPs can be conveniently fused to or derivatized with such sequences. Typically, the translocation sequence is provided as part of a fusion protein. Optionally, a linker can be used to link the ZFP and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker.
III. Position Dependence Of Subsite Recognition By Zinc Fingers
A number of the polypeptides disclosed herein have been characterized using the methods disclosed in parent application Serial No. 09/716,637 (the disclosure of which is hereby incoφorated by reference in its entirety); in particular with respect to the effect of their position, within a multi- finger protein, on their sequence specificity. The results of these investigations provide a set of zinc finger sequences that are optimized for recognition of certain triplet target subsites whose 5'-most nucleotide is a G {i.e., GNN triplet subsites). Thus, particular zinc finger sequences which recognize each of the GNN triplet subsites, from each position of a three-finger zinc finger protein, are provided. See Figure 2. It will be clear to those of skill in the art that the optimized, position- specific zinc finger sequences disclosed herein for recognition of GNN target subsites are not limited to use in three-finger proteins. For example, they are also useful in six-finger proteins, which can be made by linkage of two three-finger proteins.
A number of zinc finger amino acid sequences which are reported to bind to target subsites in which the 5 '-most nucleotide residue is G {i.e., GNN subsites) have recently been disclosed. Segal et al. (1999) Proc. Natl. Acad. Sci. USA 96:2758-2763; Drier et al. (2000) J. Mol. Biol. 303:489-502; U.S. Patent No. 6,140,081. These GNN-binding zinc fingers were obtained by selection of finger 2 sequences from phage display libraries of three-finger proteins, in which certain amino acid residues of finger 2 had been randomized. Due to the manner in which they were selected, it is not clear whether these sequences would have the same target subsite specificity if they were present in the FI and/or F3 positions.
Use of the methods and compositions disclosed herein has now allowed identification of specific zinc finger sequences that bind each of the 16 GNN triplet subsites, and for the first time, provides zinc finger sequences that are optimized for recognition of these triplet subsites in a position-dependent fashion. Moreover, in vivo studies of these optimized designs reveal that the functionality of a ZFP is correlated with its binding affinity to its target sequence. See Example 6, infra.
As a result of the discovery, disclosed herein, that sequence recognition by zinc fingers is position-dependent, it is clear that existing design rules will not, in and of themselves, be applicable to every situation in which it is necessary to construct a sequence-specific ZFP. The results disclosed herein show that many zinc fingers that are constructed based on design rules exhibit the sequence specificity predicted by those design rules only at certain finger positions. The position-specific zinc fingers disclosed herein are likely to function more efficiently in vivo and in cultured cells, with fewer nonspecific effects. Highly specific ZFPs, made using position-specific zinc fingers, will be useful tools in studying gene function and will find broad applications in areas as diverse as human therapeutics and plant engineering.
IV. Production of Zinc Finger Proteins
ZFP polypeptides and nucleic acids encoding the same can be made using routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods include Sambrook et al., Molecular Cloning, A Laboratory
Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)). In addition, nucleic acids less than about 100 bases can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA). Similarly, peptides can be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc. (http://www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio. Synthesis, Inc.
Oligonucleotides can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Nan Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either denaturing polyacrylamide gel electrophoresis or by reverse phase HPLC. The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981). Two alternative methods are typically used to create the coding sequences required to express newly designed DΝA-binding peptides. One protocol is a PCR-based assembly procedure that utilizes six overlapping oligonucleotides (Fig. 1). Three oligonucleotides (oligos 1, 3, and 5 in Figure 1) correspond to "universal" sequences that encode portions of the DΝA-binding domain between the recognition helices. These oligonucleotides typically remain constant for all zinc finger constructs. The other three "specific" oligonucleotides (oligos 2, 4, and 6 in Fig. 1) are designed to encode the recognition helices. These oligonucleotides contain substitutions primarily at positions -1, 2, 3 and 6 on the recognition helices making them specific for each of the different DNA-binding domains.
The PCR synthesis is carried out in two steps. First, a double stranded DNA template is created by combining the six oligonucleotides (three universal, three specific) in a four cycle PCR reaction with a low temperature annealing step, thereby annealing the oligonucleotides to form a DNA "scaffold." The gaps in the scaffold are filled in by high-fidelity thermostable polymerase, the combination of Taq and Pfu polymerases also suffices. In the second phase of construction, the zinc finger template is amplified by external primers designed to incoφorate restriction sites at either end for cloning into a shuttle vector or directly into an expression vector.
An alternative method of cloning the newly designed DNA-binding proteins relies on annealing complementary oligonucleotides encoding the specific regions of the desired ZFP. This particular application requires that the oligonucleotides be phosphorylated prior to the final ligation step. This is usually performed before setting up the annealing reactions. In brief, the "universal" oligonucleotides encoding the constant regions of the proteins (oligos 1 , 2 and 3 of above) are annealed with their complementary oligonucleotides. Additionally, the "specific" oligonucleotides encoding the finger recognition helices are annealed with their respective complementary oligonucleotides. These complementary oligos are designed to fill in the region which was previously filled in by polymerase in the above-mentioned protocol. The complementary oligos to the common oligos 1 and finger 3 are engineered to leave overhanging sequences specific for the restriction sites used in cloning into the vector of choice in the following step. The second assembly protocol differs from the initial protocol in the following aspects: the "scaffold" encoding the newly designed ZFP is composed entirely of synthetic DNA thereby eliminating the polymerase fill-in step, additionally the fragment to be cloned into the vector does not require amplification. Lastly, the design of leaving sequence-specific overhangs eliminates the need for restriction enzyme digests of the inserting fragment. Alternatively, changes to ZFP recognition helices can be created using conventional site-directed mutagenesis methods.
Both assembly methods require that the resulting fragment encoding the newly designed ZFP be ligated into a vector. Ultimately, the ZFP-encoding sequence is cloned into an expression vector. Expression vectors that are commonly utilized include, but are not limited to, a modified pMAL-c2 bacterial expression vector (New England BioLabs or an eukaryotic expression vector, pcDNA (Promega). The final constructs are verified by sequence analysis.
Any suitable method of protein purification known to those of skill in the art can be used to purify ZFPs (see, Ausubel, supra, Sambrook, supra). In addition, any suitable host can be used for expression, e.g., bacterial cells, insect cells, yeast cells, mammalian cells, and the like.
Expression of a zinc finger protein fused to a maltose binding protein (MBP-ZFP) in bacterial strain JM109 allows for straightforward purification through an amylose column (NEB). High expression levels of the zinc finger chimeric protein can be obtained by induction with IPTG since the MBP-ZFP fusion in the pMal-c2 expression plasmid is under the control of the tac promoter (NEB). Bacteria containing the MBP-ZFP fusion plasmids are inoculated into 2xYT medium containing lOμM ZnC12, 0.02% glucose, plus 50 μg/ml ampicillin and shaken at 37°C. At mid-exponential growth IPTG is added to 0.3 mM and the cultures are allowed to shake. After 3 hours the bacteria are harvested by centrifugation, disrupted by sonication or by passage through a french pressure cell or through the use of lysozyme, and insoluble material is removed by centrifugation. The MBP-ZFP proteins are captured on an amylose-bound resin, washed extensively with buffer containing 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5 mM DTT and 50 μM ZnC12 , then eluted with maltose in essentially the same buffer (purification is based on a standard protocol from NEB). Purified proteins are quantitated and stored for biochemical analysis.
The dissociation constants of the purified proteins, e.g., Kd, are typically characterized via electrophoretic mobility shift assays (EMSA) (Buratowski & Chodosh, in Current Protocols in Molecular Biology pp. 12.2.1-12.2.7 (Ausubel ed., 1996)). Affinity is measured by titrating purified protein against a fixed amount of labeled double-stranded oligonucleotide target. The target typically comprises the natural binding site sequence flanked by the 3 bp found in the natural sequence and additional, constant flanking sequences. The natural binding site is typically 9 bp for a three-finger protein and 2 x 9 bp + intervening bases for a six finger ZFP. The annealed oligonucleotide targets possess a 1 base 5 ' overhang which allows for efficient labeling of the target with T4 phage polynucleotide kinase. For the assay the target is added at a concentration of 1 nM or lower (the actual concentration is kept at least 10-fold lower than the expected dissociation constant), purified ZFPs are added at various concentrations, and the reaction is allowed to equilibrate for at least 45 min. In addition the reaction mixture also contains 10 mM Tris (pH 7.5), 100 mM KC1, 1 mM MgC12, 0.1 mM ZnC12, 5 mM DTT, 10% glycerol, 0.02% BSA. (NB: in earlier assays poly d(IC) was also added at 10-100 μg/μl.)
The equilibrated reactions are loaded onto a 10% polyacrylamide gel, which has been pre-run for 45 min in Tris/glycine buffer, then bound and unbound labeled target is resolved by electrophoresis at 150V. (alternatively, 10-20% gradient Tris-HCl gels, containing a 4% polyacrylamide stacker, can be used) The dried gels are visualized by autoradiography or phosphorimaging and the apparent Kd is determined by calculating the protein concentration that gives half-maximal binding.
The assays can also include determining active fractions in the protein preparations. Active fractions are determined by stoichiometric gel shifts where proteins are titrated against a high concentration of target DNA. Titrations are done at 100, 50, and 25% of target (usually at micromolar levels).
V. Applications of Engineered Zinc Finger Proteins ZPFs that bind to a particular target gene, and the nucleic acids encoding them, can be used for a variety of applications. These applications include therapeutic methods in which a ZFP or a nucleic acid encoding it is administered to a subject and used to modulate the expression of a target gene within the subject. See, for example, co-owned WO 00/41566. The modulation can be in the form of repression, for example, when the target gene resides in a pathological infecting microrganisms, or in an endogenous gene of the patient, such as an oncogene or viral receptor, that is contributing to a disease state. Alternatively, the modulation can be in the form of activation when activation of expression or increased expression of an endogenous cellular gene can ameliorate a diseased state. For such applications, ZFPs, or more typically, nucleic acids encoding them are formulated with a pharmaceutically acceptable carrier as a pharmaceutical composition.
Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition, {see, e.g., Remington 's Pharmaceutical Sciences, 17th ed. 1985)). The ZFPs, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Formulations suitable for parenteral administration, such as, for example, by intravenous, intramuscular, intradermal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
The dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose is determined by the efficacy and Kd of the particular ZFP employed, the target cell, and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also is determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular patient In other applications, ZFPs are used in diagnostic methods for sequence specific detection of target nucleic acid in a sample. For example, ZFPs can be used to detect variant alleles associated with a disease or phenotype in patient samples. As an example, ZFPs can be used to detect the presence of particular mRNA species or cDNA in a complex mixtures of mRNAs or cDNAs. As a further example, ZFPs can be used to quantify copy number of a gene in a sample. For example, detection of loss of one copy of a p53 gene in a clinical sample is an indicator of susceptibility to cancer. In a further example, ZFPs are used to detect the presence of pathological microorganisms in clinical samples. This is achieved by using one or more ZFPs specific to genes within the microorganism to be detected. A suitable format for performing diagnostic assays employs ZFPs linked to a domain that allows immobilization of the ZFP on an ELISA plate. The immobilized ZFP is contacted with a sample suspected of containing a target nucleic acid under conditions in which binding can occur. Typically, nucleic acids in the sample are labeled (e.g., in the course of PCR amplification).
Alternatively, unlabelled probes can be detected using a second labelled probe. After washing, bound-labelled nucleic acids are detected.
ZFPs also can be used for assays to determine the phenotype and function of gene expression. Current methodologies for determination of gene function rely primarily upon either overexpression or removing (knocking out completely) the gene of interest from its natural biological setting and observing the effects. The phenotypic effects observed indicate the role of the gene in the biological system.
One advantage of ZFP-mediated regulation of a gene relative to conventional knockout analysis is that expression of the ZFP can be placed under small molecule control. By controlling expression levels of the ZFPs, one can in turn control the expression levels of a gene regulated by the ZFP to determine what degree of repression or stimulation of expression is required to achieve a given phenotypic or biochemical effect. This approach has particular value for drug development. By putting the ZFP under small molecule control, problems of embryonic lethality and developmental compensation can be avoided by switching on the ZFP repressor at a later stage in mouse development and observing the effects in the adult animal. Transgenic mice having target genes regulated by a ZFP can be produced by integration of the nucleic acid encoding the ZFP at any site in trans to the target gene. Accordingly, homologous recombination is not required for integration of the nucleic acid. Further, because the ZFP is trans- dominant, only one chromosomal copy is needed and therefore functional knockout animals can be produced without backcrossing. All references cited above are hereby incoφorated by reference in their entirety for all puφoses.
EXAMPLES Example 1: Initial design of zinc finger proteins and determination of binding affinity
Initial ZFP designs were based on existing design rules, correspondence regimes and ZFP directories, including those disclosed herein {see Tables 1-5) and also in WO 98/53058; WO 98/530059; WO 98/53060 and co-owned US patent application Serial No. 09/444,241. See also WO 00/42219. Amino acid sequences were conceptually designed using amino acids 532-624 of the human transcription factor Spl as a backbone. Polynucleotides encoding designed ZFPs were assembled using a Polymerase Chain Reaction (PCR)-based procedure that utilizes six overlapping oligonucleotides. PCR products were directly cloned cloning into the Tac promoter vector, pMal-c2 (New England Biolabs, Beverly, MA) using the Kpnl and BamHI restriction sites. The encoded maltose binding protein-ZFP fusion polypeptides were purified according to the manufacturer's procedures (New England Biolabs, Beverly, MA). Binding affinity was measured by gel mobility-shift analysis. All of these procedures are described in detail in co-owned WO 00/41566 and WO 00/42219, as well as in Zhang et al. (2000) J. Biol. Chem. 275:33,850-33,860 and Liu et al. (2001) J. Biol. Chem. 276:11,323- 11,334; the disclosures of which are hereby incoφorated by reference in their entireties.
Example 2: Optimization of binding specificity by site selection
Designed ZFPs were tested for binding specificity using site selection methods disclosed in parent application USSN 09/716,637. Briefly, designed proteins were incubated with a population of labeled, double-stranded oligonucleotides comprising a library of all possible 9- or 10-nucleotide target sequences. Five nanomoles of labeled oligonucleotides were incubated with protein, at a protein concentration 4-fold above its K for its target sequence. The mixture was subjected to gel electrophoresis, and bound oligonucleotides were identified by mobility shift, and extracted from the gel. The purified bound oligonucleotides were amplified, and the amplification products were used for a subsequent round of selection. At each round of selection, the protein concentration was decreased by 2 fold. After 3-5 rounds of selection, amplification products were cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, CA), and the nucleotide sequences of approximately 20 clones were determined. The identities of the target sites bound by a designed protein were determined from the sequences and expressed as a compilation of subsite binding sequences.
Example 3: Comparison of site selection results with binding affinity To test the correlation between site selection results and the affinity of binding of a ZFP to various related targets, site selection experiments were conducted on 2 three-finger ZFPs, denoted ZFPl and ZFP2, and the site selection results were compared with Kd measurements obtained from quantitative gel- mobility shift assays using the same ZFPs and target sites. Each ZFP was constructed, based on design rules, to bind to a particular nine-nucleotide target sequence (comprising 3 three-nucleotide subsites), as shown in Figure 1. Site selection results and affinity measurements are also shown in Figure 1. The site selection results showed that fingers 1 and 3 of both the ZFPl and ZFP2 proteins preferentially selected their intended target sequences. However, the second finger of each ZFP preferentially selected subsites other than those to which they were designed to bind {e.g., F2 of ZFPl was designed to bind TCG, but preferentially selected GTG; F2 of ZFP2 was designed to bind GGT, but preferentially selected GGA).
To confirm the site selection results, binding affinities of ZFPl and ZFP2 were measured (see Example 1 , supra), both to their original target sequences and to new target sequences reflecting the site selection results. For example, the Mt- 1 sequence contains two base changes (compared to the original target sequence for ZFPl) which result in a change in the sequence of the finger 2 subsite to GTG, reflecting the preferred finger 2 subsite sequence obtained by site selection. In agreement with the site selection results, binding of ZFPl to the Mt-1 sequence is approximately 4-fold stronger than its binding to the original target sequence (Kd of 12.5 nM compared to a Kd of 50 nM, see Figure 1).
For ZFP2, the specificity of finger 2 for the 3' base of its target subsite was tested, since, although this finger was designed to bind GGT, site selection indicated that it bound preferentially to GGA. Moreover, the site selection results predicted that finger 2 of ZFP2 would bind with approximately equal affinity to GGT and GGC. Accordingly, target sequences containing GGA (Mt-3) and GGC (Mt-4) at the finger 2 subsite were constructed, and binding affinities of ZFP2 to these target sequences, and to its original target sequence (containing GGT at the finger 2 subsite), were compared. In complete agreement with the site selection results, ZFP2 exhibited the strongest binding affinity for the target sequence containing GGA at the finger 2 subsite (Kd of 0.5 nM, Figure 1), and its affinity for target sequences containing either GGT or GGC at the finger 2 subsite was approximately equal (Kd of 1 nM for both targets, Figure 1). Accordingly, the site selection method, in addition to being useful for iterative optimization of binding specificity, can also be used as a useful indicator of binding affinity.
Example 4: Use of site selection to identify position-dependent, GNN- binding zinc fingers
A large number of engineered ZFPs have been evaluated, by site selection, to identify zinc fingers that bind to GNN target subsites. In the course of these studies, it became apparent that the binding specificity of a particular zinc finger sequence is, in some instances, dependent upon the position of the zinc finger in the protein, and hence upon the location of the target subsite within the target sequence. For example, if one wishes to design a three-finger zinc finger protein to bind to a target sequence containing the triplet subsite GAT, it is necessary to know whether this subsite is the first, second or third subsite in the target sequence {i.e., whether the GAT subsite will be bound by the first, second or third finger of the protein). Accordingly, over 110 three-finger zinc finger proteins, containing potential GNN-recognizing zinc fingers in various locations, have been evaluated by site selection experiments. Generally, several zinc finger sequences were designed to recognize each GNN triplet, and each design was tested in each of the FI, F2 and F3 positions through 4 to 6 rounds of selection. The results of these analyses, shown in Figure 2, provide optimal position- dependent zinc finger sequences (the sequences shown represent amino acid residues -1 through +6 of the recognition helix portion of the finger) for recognition of the 16 GNN target subsites, as well as site selection results for these GNN-specific zinc fingers. Optimal amino acid sequences for recognition of each GNN subsite from each of three positions (finger 1, finger 2 or finger 3) are thereby provided.
GNG-binding finger designs
The amino acid sequence RSDXLXR (position -1 to +6 of the recognition helix) was found to be optimal for binding to the four GNG triplets, with Asn+3 specifying A as the middle nucleotide; His+3 specifying G as the middle nucleotide; Ala+3 specifying T as the middle nucleotide; and Asp+3 specifying cytosine as the middle nucleotide. At the +5 position, Ala, Thr, Ser, and Gin, were tested, and all showed similar specificity profiles by site selection. Interestingly, and in contrast to a previous report (Swirnoff et al. (1995) Mol. Cell. Biol. 15:2275-2287), site selection results indicated that three naturally-occurring GCG-binding fingers from zif268 and Spl, having the amino acid sequences RSDELTR, RSDELQR, and RSDERKR, were not GCG-specific. Rather, each of these fingers selected almost equal numbers of GCG and GTG sequences. Analysis of binding affinity by gel-shift experiments confirmed that finger 3 of zif268, having the sequence RSDERKR, binds GCG and GTG with approximately equal affinity.
Position dependence ofGCA-. GAT-. GGT-. GAA- and GCC-binding fingers Based on existing design rules, the amino acid sequence QSGDLTR (-1 through +6) was tested for its ability to bind the GCA triplet from three positions (FI, F2, and F3) within a three-finger ZFP. Figure 3 A shows that the QSGDLTR sequence bound preferentially to the GCA triplet subsite from the F2 and F3 positions, but not from FI. In fact, the presence of QSGDLTR at the FI position of three different three-finger ZFPs resulted predominantly in selection of GCT. Accordingly, an attempt was made to redesign this sequence to obtain specificity for GCA from the FI position. Since the sequence Q"'G+2S+3R+6 had previously been selected from a randomized FI library using GCA as target (Rebar et al. (1994) Science 263:671-673), a D (asp) to S (ser) change was made at the +3 residue of this finger. The resulting sequence, QSGSLTR, was tested for its binding specificity by site selection and found to preferentially bind GCA, from the FI position, in three different ZFPs (see Figure 2).
The QSGSLTR zinc finger, optimized for recognition of the GCA subsite from the FI position, was tested for its selectivity when located at the F2 position. Accordingly, two ZFPs, one containing QSGSLTR at finger 2 and one containing QSGDLTR at finger 2 (both having identical FI sequences and identical F3 sequences) were tested by site selection. The results indicated that, when used at the F2 position, QSGSLTR bound preferentially to GTA, rather than GCA. Thus, for optimal binding of a GCA triplet subsite from the FI position, the amino acid sequence QSGSLTR is required; while, for optimal binding of the same subsite sequence from F2 or F3, QSGDLTR should be used. Accordingly, different zinc finger amino acid sequences may be needed to specify a particular triplet subsite sequence, depending upon the location of the subsite within the target sequence and, hence, upon the position of the finger in the protein.
Positional effects were also observed for zinc fingers recognizing GAT and GGT subsites. The zinc finger amino acid sequence QSSNLAR (-1 through +6) is expected to bind to GAT, based on design rules. However, this sequence selected GAT only from the FI position, and not from the F2 and F3 positions, from which the sequence GAA was preferentially bound (Figure 3B). Similarly, the amino acid sequence QSSHLTR which, based on design rules, should bind GGT, selected GGT at the FI position, but not at the F2 and F3 positions, from which it preferentially bound GGA (Figure 3C). Conversely, the amino acid sequence TSGHLVR has previously been disclosed to recognize the triplet GGT, based on its selection from a randomized library of zif268 finger 2. U.S. Patent No. 6,140,081. However, TSGHLVR was not specific for the GGT subsite when located at the FI position (Figure 3C). These results indicate that the binding specificity of many fingers is position dependent, and particularly point out that the sequence specificity of a zinc finger selected from a F2 library may be positionally limited.
The results shown in Figure 2 indicate that recognition of at least GAA and GCC triplets by zinc fingers is also position dependent.
These positional dependences stand in contrast to earlier published work, which suggested that zinc fingers behaved as independent modules with respect to the sequence specificity of their binding to DNA. Desjarlais et al. (1993) Proc. Natl. Acad. Sci. USA 90:2256-2260. Example 5: Characterization of EP2C
The engineered zinc finger protein EP2C binds to a target sequence, GCGGTGGCT with a dissociation constant (Kd) of 2 nM. Site selection results indicated that fingers 1 and 2 are highly specific for their target subsites, while finger 3 selects GCG (its intended target subsite) and GTG at approximately equal frequencies (Figure 4A). To confirm these observations, the binding affinities of EP2C to its cognate target sequence, and to variant target sequences, was measured by standard gel-shift analyses (see Example 1 , supra). As standards for comparison, the binding affinities of Spl and zif268 to their respective targets were also measured under the same conditions, and were determined to be 40 nM for SP1 (target sequence GGGGCGGGG) and 2 nM for zif268 (target sequence GCGTGGGCG). Measurements of binding affinities confirmed that F3 of EP2C bound GTG and GCG equally well (Kds of 2 nM), but bound GAG with a twofold lower affinity (Figure 4B). Finger 2 was very specific for the GTG triplet, binding 15-fold less tightly to a GGG triplet (compare 2C0 and 2C3 in Figure 4B). Finger 1 was also very specific for the GCT triplet, it bound with 4-fold lower affinity to a GAT triplet (2C4) and with 2-fold lower affinity to a GCG triplet (2C5). This example shows, once again, the high degree of correlation between site selection results and binding affinities.
Example 6: Evaluation of engineered ZFPs by in vivo functional assays To determine whether a correlation exists between the binding affinity of a engineered ZFP to its target sequence and its functionality in vivo, cell-based reporter gene assays were used to analyze the functional properties of the engineered ZFP EP2C (see Example 5, supra). For these assays, a plasmid encoding the EP2C ZFP, fused to a VP16 transcriptional activation domain, was used to construct a stable cell line (T-Rex-293™, Invitrogen, Carlsbad, CA) in which expression of EP2C-VP16 is inducible, as described in Zhang et al, supra. To generate reporter constructs, three tandem copies of the EP2C target site, or its variants (see Figure 4B, column 2), were inserted between the Mlu I and Bglll sites of the pGL3 lucifεrase-encoding vector (Promega, Madison, WI), upstream of the SV40 promoter. Structures of all reporter constructs were confirmed by DNA sequencing. Luciferase reporter assays were performed by co-transfection of luciferase reporter construct (200 ng) and pCMV- βgal (100 ng, used as an internal control) into the EP2C cells seeded in 6-well plates. Expression of the EP2C-VP16 transcriptional activator was induced with doxycycline (0.05 ug/ml) 24 h after transfection of reporter constructs. Cell lysates were harvested 40 hours post- transfection, luciferase and β-galactosidase activities were measured by the Dual- Light Reporter Assay System (Tropix, Bedford, MA), and luciferase activities were normalized to the co-transfected β-galactosidase activities. The results, shown on the right side of Figure 4B, showed that the normalized luciferase activity for each reporter construct was well correlated with the in vitro binding affinity of EP2C to the target sequence present in the construct. For example, the target sequences to which EP2C bound with greatest affinity (2C0 and 2C2, Ka of 2 nM for each) both stimulated the highest levels of luciferase activity, when used to drive luciferase expression in the reporter construct (Figure 4B). Target sequences to which EP2C bound with 2-fold lower affinity, 2C1 and 2C5 (Kd of 4 nM for each), stimulated roughly half the luciferase activity of the 2C0 and 2C2 targets. The 2C3 and 2C4 sequences, for which EP2C showed the lowest in vitro binding affinities, also yielded the lowest levels of in vivo activity when used to drive luciferase expression. Target 3B, a sequence to which EP2C does not bind, yielded background levels of luciferase activity, similar to those obtained with a luciferase-encoding vector lacking EP2C target sequences (pGL3). Thus there exist good correlations between binding affinity (as determined by Kd measurement), binding specificity (as determined by site selection) and in vivo functionality for engineered zinc finger proteins.
TABLE 1
SEQ SEQ SEQ SEQ Kd
SBS# TARGET ID FI ID F2 ID ___ ID (nM) 249 GCGGGGGCG 17 RSDELTR 123 RSDHLSR 229 RSDELRR 335 20 250 GCGGGGGCG 18 RSDELTR 124 RSDHLSR 230 RSDTLKK 336 70 251 GCGGAGGCG 19 RSDELTR 125 RSDNLTR 231 RSDELRR 337 27.5 252 GCGGCCGCG 20 RSDELTR 126 DRSSLTR 232 RSDELRR 338 100 253 GGATGGGGG 21 RSDHLAR 127 RSDHLTT 233 QRAHLAR 339 0.75 256 GCGGGGTCC 22 ERGDLTT 128 RSDHLSR 234 RSDELRR 340 800 258 GCGGGCGGG 23 RSDHLTR 129 ERGHLTR 235 RSDELRR 341 15 259 GCAGAGGAG 24 RSDNLAR 130 RSDNLAR 236 QSGSLTR 342 250 261 GAGGTGGCC 25 ERGTLAR 131 RSDALSR 237 RSDNLSR 343 0.5 262 GCGGGGGCT 26 QSSDLQR 132 , RSDHLSR 238 RSDELRR 344 20 263 GCGGGGGCT 27 QSSDLQR 133 RSDHLSR 239 RSDTLKK 345 1 264 GTGGCTGCC 28 DRSSLTR 134 QSSDLQR 240 RSDALAR 346 27 265 GTGGCTGCC 29 ERGTLAR 135 QSSDLQR 241 RSDALAR 347 600
269 GGGGCCGGG 30 RSDHLTR 136 DRSSLTR 242 RSDHLTR 348 5
270 GGGGCCGGG 31 RSDHLTR 137 ERGTLAR 243 RSDHLTR 349 52.5
272 GCAGGGGCC 32 DRSSLTR 138 ' RSDHLSR 244 QSGSLTR 350 20
337 TGCGGGGCAA 33 RSADLTR 139 RSDHLTR 245 ERQHLAT 351 24 338 TGCGGGGCAA 34 RSADLTR 140 RSDHLTR 246 ERDHLRT 352 8 339 TGCGGGGCAA 35 RSADLTR 141 RSDHLTT 247 ERQHLAT 353 64 340 TGCGGGGCAA 36 RSADLTR 142 RSDHLTT 248 ERDHLRT 354 48 341 TGCGGGGCAA 37 RSADLTR 143 RGDHLKD 249 ERQHLAT 355 1000 342 TGCGGGGCAA 38 RSADLTR 144 RGDHLKD 250 ERDHLRT 356 1000 343 TGCGGGGCAA 39 QSGSLTR 145 RSDHLTR 251 ERQHLAT 357 8 344 TGCGGGGCAA 40 QSGSLTR 146 RSDHLTR 252 ERDHLRT 358 6 345 TGCGGGGCAA 41 QSGSLTR 147 RSDHLTT 253 ERQHLAT 359 96 346 TGCGGGGCAA 42 QSGSLTR 148 RSDHLTT 254 ERDHLRT 360 64 347 TGCGGGGCAA 43 QSGSLTR 149 " RGDHLKD 255 ERQHLAT 361 1000 348 TGCGGGGCAA 44 QSGSLTR 150 RGDHLKD 256 ERDHLRT 362 1000
367 GGGGGCGGG 45 RSDHLTR 151 DSGHLTR 257 RSDHLQR 363 60
368 GAGGGGGCG 46 RSDELTR 152 RSDHLTR 258 RSDNLTR 364 3.5
369 GTAGTTGTG 47 RSDALTR 153 TGGSLAR 259 QSGSLTR 365 95
370 GTAGTTGTG 48 RSDALTR 154 NRATLAR 260 QSASLTR 366 300
371 GTAGTTGTG 49 RSDALTR 155 NRATLAR 261 QSGSLTR 367 175
372 GTAGTTGTG 50 RSDSLLR 156 TGGSLAR 262 QSASLTR 368 112.5
373 GTAGTTGTG 51 RSDSLLR 157 NRATLAR 263 QSASLTR 369 320
374 GCTGAGGAA 52 QRSNLVR 158 RSDNLTR 264 TSSELQR 370 3.3
375 GAGGAAGAT 53 QQSNLAR 159 QSGNLQR 265 RSDNLTR 371 85
401 GTAGTTGTG 54 RSDALTR 160 TGGSLAR 266 QSASLTR 372 80 403 GTAGTTGTG 55 RSDSLLR 161 NRATLAR 267 QSGSLTR 373 750
421 GTAGTTGTG 56 DSDSLLR 162 TGGSLAR 268 QSGSLTR 374 500
422 GTAGTTGTG 57 RSDSLLR 163 TGGSLTR 269 QSGSLTR 375 200
423 GTAGTTGTG 58 RSDALTR 164 TGGSLAR 270 QRSALAR 376 1000
424 GATGCTGAG 59 RSDNLTR 165 TSSELQR 271 TSANLSR 377 100
425 GATGCTGAG 60 RSDNLTR 166 QSSDLQR 272 QQSNLAR 378 25 426 GATGCTGAG 61 RSDNLTR 167 QSSDLQR 273 TSANLSR 379 5.5 427 GCTGAGGAA 62 QRSNLVR 168 RSDNLTR 274 QSSDLQR 380 1 428 GAAGATGAC 63 DSSNLTR 169 QQSNLAR 275 QRSNLVR 381 120
429 GAAGATGAC 64 DSΞNLTR 170 TSANLSR 276 QRSNLVR 382 50
430 GATGACGAC 65 EKANLTR 171 DSSNLTR 277 QQSNLAR 383 250 431 GACGACGGC 66 DSGHLTR 172 DRSNLER 278 DSSNLTR 384 100 432 GACGACGGC 67 DSGHLTR 173 DHANLAR 279 DSSNLTR 385 1000 433 GACGACGGC 68 DSGNLTR 174 DHANLAR 280 DSSNLTR 386 1000 434 GACGGCGTA 69 QSASLTR 175 DSGHLTR 281 EKANLTR 387 152.5 435 GACGGCGTA 70 QSASLTR 176 . DSGHLTR 282 ERGNLTR 388 150 436 GACGGCGTA 71 QRSALAR 177 DSGHLTR 283 EKANLTR 389 95 437 GACGGCGTA 72 QRSALAR 178 DSGHLTR 284 ERGNLTR 390 117.5 438 GAGGGGGCG 73 RSDELTR 179 RSDHLTT 285 RSDNLTR 391 62.5
440 GCCGAGGTGC 74 RSDSLLR 180 RSKNLQR 286 ERGTLAR 392 40
441 GGTGGAGTCA 75 DSGSLTR 181 QSGHLQR 287 TSGHLTR 393 250 445 GTCGCAGTGA 76 RSDSLRR 182 " QSSDLQK288 DSGSLTR 394 1000 450 GACTTGGTGC 77 RSDTLAR 183 RGDALTS 289 DRSNLTR 395 130 453 GGTGGAGTCA 78 DRSALAR 184 QSGHLQR 290 DSSKLSR 396 150
461 GAGTACTGTA 79 QRSHLTT 185 DRSNLRT 291 RSDNLAR 397 120
463 GTGGAGGAGA 80 RSDNLTR 186 RSDNLAR 292 RSDALAR 398 0.5
464 GTGGAGGAGA 81 RSDNLTR 187 ' ' RSDNLAR 293 RSDSLAR 399 0.4
466 CAGGCTGCGC 82 RSDDLTR 188 QSSDLQR 294 RSDNLRE 400 65
467 CAGGCTGCGC 83 RSDELTR 189 QSSDLQR 295 RGDHLKD 401 800
468 CAGGCTGCGC 84 RSDDLTR 190 QSSDLQR 296. RGDHLKD 402 42
469 GAAGAGGTCT 85 DRSALAR 191 RSDNLAR 297 QSGNLTR 403 13.5
472 GAGGTCTGGA 86 RSSHLTT 192 DRSALAR 298 RSDNLAR 404 80
476 GGAGAGGATG 87 TTSNLRR 193 RSDNLAR 299 QSDHLTR 405 80
A l l GGAGAGGATG 88 TTSNLRR 194 RSDNLAR 300 QRAHLAR 406 100
478 GGAGAGGATG 89 TTSNLRR 195 RSDNLAR 301 QSGHLRR 407 60
479 GTGGCGGACC 90 DSSNLTR 196 RSDELQR 302 RSDALAR 408 8.5
480 GTGGCGGACC 91 DSSNLTR 197 RADTLRR 303 RSDALAR 409 5
483 GAGGGCGAAG 92 QSANLAR 198 " ESSKLKR 304 RSDNLAR 410 130
484 GAGGGCGAAG 93 QSDNLAR 199 ESSKLKR 305 RSDNLAR 411 1000
485 GGAGAGGTTT 94 QSSALAR 200 RSDNLAR 306 QRAHLAR 412 110
487 GGAGAGGTTT 95 NRATLAR 201 RSDNLAR 307 QSGHLAR 413 76.9
488 TGGTAGGGGG 96 RSDHLAR 202 RSDNLTT 308 RSDHLTT 414 35
490 TAGGGGGTGG 97 RSDSLLR 203 RSDHLTR 309 RSDNLTT 415 1.5
503 GCCGAGGTGC 98 RSDSLLR 204 RSDNLAR 310 ERGTLAR 416 50
504 GCCGAGGTGC 99 RSDSLLR 205 RSDNLAR 311 DRSDLTR 417 25
505 GCCGAGGTGC 100 RSDSLLR 206 RSDNLAR 312 DCRDLAR 418 65
526 GCGGGCGGGC 101 RSDHLTR 207 ERGHLTR 313 RSDTLKK 419 8
543 GAGTGTGTGA 102 RSDLLQR 208 MSHHLKE 314 RSDHLSR 420 50
544 GAGTGTGTGA 103 RSDSLLR 209 • MSHHLKE 315 RSDNLAR 421 125
545 GAGTGTGTGA 104 RKDSLVR 210 TSDHLAS 316 RSDNLTR 422 32
546 GAGTGTGTGA 105 RSDLLQR 211 MSHHLKT 317 RLDGLRT 423 500
547 GAGTGTGTGA 106 RKDSLVR 212 TSGHLTS 318 RSDNLTR 424 500
548 GAGTGTGTGA 107 RSSLLQR 213 MSHHLKT 319 RSDHLSR 425 500
549 GAGTGTGTGA 108 RSSLLQR 214 MSHHLKE 320 RSDHLSR 426 500
550 GAGTGTGTGA 109 RKDSLVR 215 TKDHLAS 321 RSDNLTR 427 20
551 GAGTGTGTGA 110 RSDLLQR 216 MSHHLKT 322 RSDHLSR 428 50
552 GAGTGTGTGA 111 RKDSLVR 217 MSHHLKT 323 RSDNLTR 429 31 553 GAGTGTGTGA 112 RSDSLLR 218 MSHHLKE 324 RSDNLTR 430 125
554 GAGTGTGTGA 113 RKDSLVR 219 TSDHLAS 325 RSDNLAR 431 62.5
558 TGCGGGGCA 114 QSGDLTR 220 RSDHLTR 326 DSGHLAS 432 21
559 GAGTGTGTGA 115 RSDSLLR 221 TSDHLAS 327 RSDNLAR 433 1000
560 GAGTGTGTGA 116 RSSLLQR 222 MSHHLKT 328 RSDHLSR 434 500
561 GAGTGTGTGA 117 RKDSLVR 223 MSHHLKE 329 RSDNLAR 435 1000
562 GAGTGTGTGA 118 RSDSLLR 224 TSGHLTS 330 RSDNLAR 436 1000 565 GATGCTGAG 119 RSDNLTR 225 TSSELQR 331 QQSNLAR 437 100
567 GAAGATGAC 120 EKANLTR 226 TSANLSR 332 QRSNLVR 438 47.5
568 GATGACGAC 121 EKANLTR 227 DSSNLTR 333 TSANLSR 439 300
569 GTAGTTGTG 122 RSDSLLR 228 TGGSLAR 334 QRSALTR 440 52
TABLE 2
SEQ SEQ SEQ SEQ Kd
SBS# TARGET ID FI ID F2 ID F3 ID (nM)
201 GCAGCCTTG 441 RSDSLTS 646 ERSTLTR 851 QRADLRR 1056 1000
202 GCAGCCTTG 442 RSDSLTS 647 ERSTLTR 852 QRADLAR 1057 1000
203 GCAGCCTTG 443 RSDSLTS 648 ERSTLTR 853 QRATLRR 1058 1000
204 GCAGCCTTG 444 RSDSLTS 649 ERSTLTR 854 QRATLAR 1059 1000 205 GAGGTAGAA 445 QSANLAR 650 QSATLAR 855 RSDNLSR 1060 80 206 GAGGTAGAA 446 QSANLAR 651 QSAVLAR 856 RSDNLSR 1061 1000 207 GAGTGGTTA 447 QRASLAS 652 RSDHLTT 857 RSDNLAR 1062 70
208 TAGGTCTTA 448 QRASLAS 653 DRSALAR 858 RSDNLAS 1063 1000
209 GGAGTGGTT 449 QSSALAR 654 RSDALAR 859 QRAHLAR 1064 35
210 GGAGTGGTT 450 NRDTLAR 655 RSDALAR 860 QRAHLAR 1065 65
211 GGAGTGGTT 451 QSSALAR 656 RSDALAS 861 QRAHLAR 1066 140
212 GGAGTGGTT 452 NRDTLAR 657 RSDALAS 862 QRAHLAR 1067 400
213 GTTGCTGGA 453 QRAHLAR 658 QSSTLAR 863 QSSALAR 1068 1000
214 GTTGCTGGA 454 QRAHLAR 659 QSSTLAR 864 NRDTLAR 1069 1000
215 GAAGTCTGT 455 NRDHLMV 660 DRSALAR 865 QSANLSR 1070 1000
216 GAAGTCTGT 456 NRDHLTT 661 DRSALAR 866 QSANLSR 1071 1000
217 GAGGTCGTA 457 QRSALAR 662 DRSALAR 867 RSDNLAR 1072 40
219 GATGTTGAT 458 QQSNLAR 663 NRDTLAR 868 NRDNLSR 1073 1000
220 GATGTTGAT 459 QQSNLAR 664 NRDTLAR 869 QQSNLSR 1074 1000
221 GATGAGTAC 460 DRSNLRT 665 RSDNLAR 870 NRDNLAR 1075 1000 222 GATGAGTAC 461 ERSNLRT 666 RSDNLAR 871 NRDNLAR 1076 1000
223 GATGAGTAC 462 DRSNLRT 667 RSDNLAR 872 QQSNLAR 1077 105
224 GATGAGTAC 463 ERSNLRT 668 RSDNLAR 873 QQSNLAR 1078 1000
225 TGGGAGGTC 464 DRSALAR 669 .RSDNLAR 874 RSDHLTT 1079 6
226 GCAGCCTTG 465 RGDALTS 670 ERGTLAR 875 QSGSLTR 1080 1000
227 GCAGCCTTG 466 RGDALTV 671 ERGTLAR 876 QSGSLTR 1081 1000
228 GCAGCCTTG 467 RGDALTM 672 ERGTLAR 877 QSGSLTR 1082 1000
229 GCAGCCTTG 468 RGDALTS 673 ERGTLAR 878 RSDELTR 1083 1000
230 GCAGCCTTG 469 RGDALTV 674 ERGTLAR 879 RSDELTR 1084 1000 231 GCAGCCTTG 470 RGDALTM 675 ERGTLAR 880 RSDELTR 1085 1000
232 GGTGTGGTG 471 RSDALTR 676 RSDALAR 881 NRSHLAR 1086 50
233 GGTGTGGTG 472 RSDALTR 677 RSDALAR 882 QASHLAR 1087 100
235 GTAGAGGTG 473 RSDALTR 678 RSDNLAR 883 QRGALAR 1088 80
236 GGGGAGGGG 474 RSDHLAR 679 RSDNLAR 884 RSDHLSR 1089 0.3
237 GGGGAGGCC 475 ERGTLAR 680 RSDNLAR 885 RSDHLSR 1090 0.3
238 GGGGAGGCC 476 ERGTLAR 681 RSDNLQR 886 RSDHLSR 1091 0.8
239 GGCGGGGAG 477 RSDNLTR 682 RSDHLTR 887 DRSHLAR 1092 0.4
240 GCAGGGGAG 478 RSDNLTR 683 RSDHLSR 888 QSGSLTR 1093 1
242 GGGGGTGCT 479 QSSDLRR 684 QSSHLAR 889 RSDHLSR 1094 1
243 GTGGGCGCT 480 QSSDLRR 685 DRSHLAR 890 RSDALAR 1095 75
244 TAAGAAGGG 481 RSDHLAR 686 QSGNLTR891 QSGNLRT 1096 100
245 TAAGAAGGG 482 RSDHLAR 687 QSANLTR 892 QSGNLRT 1097 235
246 GAAGGGGAG 483 RSDNLAR 688 RSDHLAR 893 QSGNLTR 1098 2
247 GAAGGGGAG 484 RSDNLAR 689 RSDHLAR 894 QSGNLRR 1099 2 276 GCGGCCGCG 485 RSDELTR 690 ERGTLAR 895 RSDERKR 1100 90
277 GCGGCCGCG 486 RSDELTR 691 'DRSSLTR 896 RSDERKR 1101 107
278 GCGGCCGCG 487 QS ELTR 692 ERGTLAR 897 RSDERKR 1102 190
279 GCGGCCGCG 488 QSWELTR 693 DRSSLTR 898 RSDERKR 1103 260
280 GCGGCCGCG 489 QSGSLTR 694 ERGTLAR 899 RSDERKR 1104 160
281 GCGGCCGCG 490 QSGSLTR 695 DRSSLTR 900 RSDERKR 1105 225 282 GCAGAAGTG 491 RGDALTR 696 QSANLTR 901 QSADLAR 1106 1000
283 GCAGAAGTG 492 RSDALTR 697 QSGNLTR 902 QSGSLTR 1107 2
284 GCGGCCGCG 493 QSGSLTR 698 RSDHLTT 903 RSDERKR 1108 1000
285 TGTGCGGCC 494 ERGTLAR 699 RSDELTR 904 SRDHLQS 1109 1000
287 GCAGAAGCG 495 RGPDLAR 700 QSANLTR 905 QSGSLTR 1110 1000
288 GCAGAAGCG 496 RGPDLAR 701 QSANLTR 906 QSGSLTR 1111 1000
289 GCAGAAGCG 497 RGPDLAR 702 QSGNLQR 907 QSGSLTR 1112 800 290 GCAGAAGCG 498 RSDELAR 703 QSANLQR 908 QSADLAR 1113 1000
292 GCAGAAGCG 499 RSDELTR 704 QSANLQR 909 QSGSLTR 1114 1000
293 GTGTGCGGC 500 DRSHLTR 705 ERHSLQT 910 RSDALTR 1115 320 296 TGCGCGGCC 501 ERGTLAR 706 RSDELTR 911 DRDHLQS 1116 1000
297 TGCGCGGCC 502 ERGTLAR 707 RSDELRR 912 DRSHLQT 1117 500
298 GCTTAGGCA 503 QTGELRR 708 RSDNLQK 913 TSGDLSR 1118 4000 299 GCTTAGGCA 504 QTSDLRR 709 RSDNLQK 914 QSSDLQR 1119 4000
300 GCTTAGGCA 505 QTADLRR 710 RSDNLQR 915 QSSDLSR1120 400
301 GCTTAGGCA 506 QSADLRR 711 RSDNLQT 916 QSSDLSR1121 350
302 GCTTAGGCA 507 QSGSLTR 712 RSDNLQT 917 QSSDLSR 1122 75
303 GCTTAGGCA 508 QTGSLTR 713 RSDNLQT 918 QSSDLSR 1123 135
304 GCTTAGGCA 509 QTADLTR 714 RSDNLQT 919 QSSDLSR 1124 230
305 GCTTAGGCA 510 QTGDLTR 715 RSDNLQT 920 QSSDLSR 1125 230
306 GCTTAGGCA 511 QTASLTR 716 RSDNLQT 921 QSSDLSR 1126 280
307 GAAGAAGCG 512 RSDELRR 717 QSGNLQR 922 QSGNLSR 1127 50.5
308 GAAGAAGCG 513 RSDELRR 718 QSANLQR 923 QSANLQR 1128 1000
309 GGAGATGCC 514 ERSDLRR 719 QSSNLQR 924 QSGHLSR 1129 4000
310 GGAGATGCC 515 DRSDLTR 720 NRDNLQT 925 QSGHLSR 1130 1000
311 GGAGATGCC 516 DRSTLTR 721 NRDNLQR 926 QSGHLSR 1131 170
312 GGAGATGCC 517 ERGTLAR 722 NRDNLQR 927 QSGHLSR 1132 2000
313 GGAGATGCC 518 DRSDLTR 723 QRSNLQR 928 QSGHLSR 1133 1000
314 GGAGATGCC 519 DRSSLTR 724 QSSNLQR 929 QSGHLSR 1134 117.5
315 GGAGATGCC 520 ERGTLAR 725 QSSNLQR 930 QSGHLSR 1135 265
316 GGAGATGCC 521 ERGTLAR 726 QRDNLQR931 QSGHLSR 1136 3000
318 TAGGAGATGC 522 RSDALTS 727 RSDNLAR 932 RSDNLAS1137 100
319 GGGGAAGGG 523 KTSHLRA728 QSGNLSR 933 RSDHLSR 1138 125
320 GGGGAAGGG 524 RSDHLTR 729 .QSGNLSR 934 RSDHLSR 1139 5
321 GGCGGAGAT 525 TTSNLRR 730 QSGHLQR 935 DRSHLTR 1140 200
323 GGCGGAGAT 526 TTSNLRR 731 QSGHLQR 936 DRDHLTR 1141 600
324 GGCGGAGAT 527 TTSNLRR 732 QSGHLQR 937 DRDHLTR 1142 200
325 GTATCTGCT 528 NSSDLTR 733 NSDVLTS 938 QSDVLTR 1143 1000
326 GTATCTGTT 529 NSDALTR 734 NSDVLTS 939 QSDVLTR 1144 1000
327 TCTGCTGGG 530 RSDHLTR 735 NSADLTR 940 NSDDLTR 1145 1000
328 TCTGTTGGG 531 RSDHLTR 736 NSSALTS 941 NSDDLTR 1146 1000
349 GGTGTCGCC 532 DCRDLAR 737 DSGSLTR 942 TSGHLTR 1147 1000
350 TCCGAGGGT 533 TSGHLTR 738 RSDNLTR 943 DCRDLTT 1148 332
351 GCTGGTGTC 534 DSGSLTR 739 TSGHLTR 944 TLHTLTR1149 1000
352 GGAGGGGTG 535 RSDSLLR 740 RSDHLTR 945 QSDHLTR 1150 26
353 GTTGGAGCC 536 DCRDLAR 741 QSDHLTR 946 TSGALTR 1151 1000
354 GAAGAGGAC 537 DSSNLTR 742 RSDNLTR 947 QRSNLVR 1152 28 355 GAAGAGGAC 538 EKANLTR 743 RSDNLTR 948 QRSNLVR 1153 20
356 GGCTGGGCG 539 RSDELRR 744 RSDHLTK 949 DSDHLSR 1154 1000
357 GGCTGGGCG 540 RSDELRR 745 RSDHLTK 950 DSDHLSR 1155 1000
358 GGCTGGGCG 541 RSDELRR 746 RSDHLTK 951 DSSHLSR 1156 225 361 GGGTTTGGG 542 RSDHLTR 747 QSSALTR 952 RSDHLTR 1157 130
363 GGGTTTGGG 543 RSDHLTR 748 QSSVLTR 953 RSDHLTR 1158 200
364 GTGTCCGAAG 544 RSDNLTR 749 DSAVLTT 954 RSDSLTR 1159 1000 365 GGTGCTGGT 545 QASHLTR 750 QASVLTR 955 QASHLTR 1160 600
366 GAGGGTGCT 546 QASVLTR 751 QASHLTR 956 RSDNLTR 1161 1000
367 GGGGGCGGG 547 RSDHLTR 752 DSGHLTR 957 RSDHLQR 1162 60
368 GAGGGGGCG 548 RSDELTR 753 RSDHLTR 958 RSDNLTR 1163 3.5 369 GTAGTTGTG 549 RSDALTR 754 TGGSLAR 959 QSGSLTR 1164 95
370 GTAGTTGTG 550 RSDALTR 755 NRATLAR 960 QSASLTR 1165 300
371 GTAGTTGTG 551 RSDALTR 756 NRATLAR 961 QSGSLTR 1166 175
372 GTAGTTGTG 552 RSDSLLR 757 TGGSLAR 962 QSASLTR 1167 112.5
373 GTAGTTGTG 553 RSDSLLR 758 NRATLAR 963 QSASLTR 1168 320
374 GCTGAGGAA 554 QRSNLVR 759 RSDNLTR 964 TSSELQR 1169 3.3 375 GAGGAAGAT 555 QQSNLAR 760 QSGNLQR 965 RSDNLTR 1170 85 377 GTGTTGGCAG 556 QSGSLTR 761 RGDALTS 966 RSDALTR 1171 89 378 GCCGAGGAGA 557 RSDNLTR 762 -RSDNLTR 967 DRSSLTR 1172 31 379 GCCGAGGAGA 558 RSDNLTR 763 RSDNLTR 968 ERGTLAR 1173 3
380 GAGTCGGAAG 559 QSANLAR 764 RSDELTT 969 RSDNLAR 1174 1000
381 GCAGCTGCGC 560 RSDELTR 765 QSSDLQR 970 QSGDLTR 1175 1.5
383 TGGTTGGTAT 561 QSATLAR 766 RGDALTS 971 RSDHLTT 1176 1000
384 GTGGGCTTCA 562 DRSALTT 767 DRSHLAR 972 RSDALAR 1177 60
385 GGGGCGGAGC 563 RSDNLTR 768 RSDTLKK 973 RSDHLSR 1178 1.2
386 GGGGCGGAGC 564 RSDNLTR 769 RSDELQR 974 RSDHLSR 1179 0.4
387 GGCGAGGCAA 565 QSGSLTR 770 RSDNLAR 975 DRSHLAR 1180 2.5
388 GGCGAGGCAA 566 QSGDLTR 771 RSDNLAR 976 DRSHLAR 1181 28
390 GTGGCAGCGG 567 RSDTLKK 772 QSSDLQK 977 RSDALAR 1182 20
392 GTGGCAGCGG 568 RSDELTR 773 .QSSDLQK 978 RSDALAR 1183 1000
396 GCGGGAGCAG 569 QSGSLTR 774 QSGHLQR 979 RSDTLKK 1184 18.8
397 GCGGGAGCAG 570 QSGDLTR 775 QSGHLQR 980 RSDTLKK 1185 25
400 TCAGTGGTGG 571 RSDALAR 776 RSDSLAR 981 QSGDLRT 1186 40 405 GCGGCCGCA 572 RSDELTR 777 ERGTLAR 982 RSDERKR 1187 110
406 GCGGCCGCA 573 RSDELTR 778 .DRSSLTR 983 RSDERKR 1188 110
407 GCGGCCGCA 574 QSWELTR 779 ERGTLAR 984 RSDERKR 1189 410
408 GCGGCCGCA 575 QSWELTR 780 DRSSLTR 985 RSDERKR 1190 380
409 GCGGCCGCA 576 QSGSLTR 781 ERGTLAR 986 RSDERKR 1191 50
410 GCAGAAGTC 577 RSDALTR 782 QSGNLTR 987 QSGSLTR 1192 3
411 GCGGCCGCA 578 QSGSLTR 783 RSDHLTT 988 RSDERKR 1193 1000
412 GCGTGGGCG 579 QSGSLTR 784 RSDHLTT 989 RSDERKR 1194 5
413 GCGTGGGCA 580 QSGSLTR 785 RSDHLTT 990 RSDERKR 1195 5
414 GCAGAAGCA 581 RSDELTR 786 QSANLQR 991 QSGSLTR 1196 1000
415 GTGTGCGGA 582 DRSHLTR 787 ERHSLQT 992 RSDALTR 1197 1000
416 TGTGCGGCC 583 ERGTLAR 788 RSDELRR 993 DRSHLQT 1198 1000 493 GGGGTGGCGG 584 RSDTLKK 789 RSDSLAR 994 RSDHLSR 1199 300
494 GCCGAGGAGA 585 RSDNLTR 790 RSDNLTR 995 DRSSLTR 1200 90
496 GGTGGTGGC 586 DTSHLRR 791 TSGHLQR 996 TSGHLSR 1201 1000
497 GTTTGCGTC 587 ETASLRR 792 DSAHLQR 997 TSSALSR 1202 1000
498 GAAGAGGCA 588 QTGELRR 793 RSDNLQR 998 QSGNLSR 1203 30
499 GCTTGTGAG 589 RTSNLRR 794 TSSHLQK 999 DTDHLRR1204 1000
500 GCTTGTGAG 590 RSDNLTR 795 QSSNLQT 1000 DRSHLAR 1205 1000
501 GTGGGGGTT 591 NRATLAR 796 RSDHLSR 1001 RSDALAR 1206 8 502 GGGGTGGGA 592 QSAHLAR 797 RSDALAR 1002 RSDHLSR 1207 60 507 GAGGTAGAGG 593 RSDNLAR 798 QRSALAR 1003 RSDNLAR 1208 10
508 GAGGTAGAGG 594 RSDNLAR 799 QSATLAR 1004 RSDNLAR 1209 10
509 GTCGTGTGGC 595 RSDHLTT 800 RSDALAR 1005 DRSALAR 1210 100
510 GTTGAGGAAG 596 QSGNLAR 801 RSDNLAR 1006 NRATLAR 1211 100
511 GTTGAGGAAG 597 QSGNLAR 802 RSDNLAR 1007 QSSALAR 1212 100
512 GAGGTGGAAG 598 QSGNLAR 803 RSDALAR 1008 RSDNLAR 1213 10
513 GAGGTGGAAG 599 QSANLAR 804 RSDALAR 1009 RSDNLAR 1214 1.5
514 TAGGTGGTGG 600 RSDALTR 805 RSDALAR 1010 RSDNLTT 1215 10
515 TGGGAGGAGT 601 RSDNLTR 806 -RSDNLTR 1011 RSDHLTT 1216 0.5
516 GGAGGAGCT 602 TTSELRR 807 QSGHLQR 1012 QSGHLSR 1217 700
517 GGAGCTGGGG 603 RTDHLRR 808 TSSELQR 1013 QSGHLSR 1218 50
518 GGGGGAGGAG 604 QTGHLRR 809 QSGHLQR 1014 RSDHLSR 1219 30
519 GGGGAGGAGA 605 RSDNLAR 810 RSDNLSR 1015 RSDHLSR 1220 0.3 520 GGAGGAGAT 606 TTANLRR 811 -QSGHLQR 1016 QSGHLSR 1221 300
521 GCAGCAGGA 607 QTGHLRR 812 QSGELQR 1017 QSGELSR 1222 1000
522 GATGAGGCA 608 QTGELRR 813 RSDNLQR 1018 TSANLSR 1223 200
527 GGGGAGGATC 609 TTSNLRR 814 RSSNLQR 1019 RSDHLSR 1224 2
528 GGGGAGGATC 610 TTSNLRR 815 RSSNLQR 1020 RSDHLSR 1225 10
529 GAGGCTTGGG 611 RTDHLRK 816 TSAELQR 1021 RSSNLSR 1226 1000
531 GCGGAGGCTT 612 TTGELRR 817 RSSNLQR 1022 RSDELSR 1227 160
532 GCGGAGGCTT 613 QSSDLQR 818 RSSNLQR 1023 RSDELSR 1228 100 533 GCGGAGGCTT 614 QSSDLQR 819 RSDNLAR 1024 RSADLSR 1229 7
534 GCGGAGGCTT 615 QSSDLQR 820 RSDNLAR 1025 RSDDLRR1230 10
535 GCAGCCGGG 616 RTDHLRR 821 ESSDLQR 1026 QSGELSR 1231 1000 538 GCAGAGGCTT 617 QSSDLQR 822 -RSDNLAR 1027 QSGSLTR 1232 70
540 TGGGCAGGCC 618 DRSHLTR 823 QSGSLTR 1028 RSDHLTT 1233 55
541 GGGGAGGAT 619 TTSNLRR 824 RSSNLQR 1029 RSDHLSR 1234 3
570 GGGGAAGGCT 620 DSGHLTR 825 QRSNLVR 1030 RSDHLTR 1235 20
571 GTGTGTGTGT 621 RSDSLTR 826 QRSNLVR 1031 RSDSLLR 1236 1000 572 GCATACGTGG 622 RSDSLLR 827 DKGNLQS 1032 QSDDLTR 1237 1000
573 GCATACGTG 623 RSDSLLR 828 DKGNLQS 1033 QSGDLTR 1238 1000
574 TACGTGGGGT 624 RSDHLTR 829 RSDHLTR 1034 DKGNLQT 1239 25
575 TACGTGGGCT 625 DFSHLTR 830 RSDHLTR 1035 DKGNLQT 1240 472
576 GAGGGTGTTG 626 NSDTLAR 831 TSGHLTR 1036 RSDNLTR 1241 200
577 GGAGCGGGGA 627 RSDHLSR 832 RSDELQR1037 QSDHLTR 1242 200 579 GGGGTTGAGG 628 RSDNLTR 833 .NRDTLAR 1038 TSGHLTR 1243 200 580 GGTGTTGGAG 629 QRAHLAR 834 NRDTLAR 1039 TSGHLTR 1244 1000 581 TACGTGGGTT 630 QSSHLTR 835 RSDSLLR 1040 DKGNLQT 1245 382
583 GTAGGGGTTG 631 NSSALTR 836 RSDHLTR 1041 QSASLTR 1246 46
584 GAAGGCGGAG 632 QAGHLTR 837 DKSHLTR 1042 QSGNLTR 1247 1000
585 GAAGGCGGAG 633 QAGHLTR 838 DSGHLTR 1043 QSGNLTR 1248 1000 587 GGGGGTTACG 634 DKGNLQT 839 TSGHLTR 1044 RSDHLSK1249 500 588 GGGGGGGGGG 635 RSDHLSR 840 RSDHLTR 1045 RSDHLSK 1250 30 589 GGAGTATGCT 636 DSGHLAS 841 QSATLAR 1046 QSDHLTR 1251 1000 595 TGGTTGGTAT 637 QRGSLAR 842 RGDALTR 1047 RSDHLTT 1252 73.3
597 TGGTTGGTA 638 QNΞAMRK 843 RGDALTS 1048 RSDHLTT 1253 1000
598 TGGTTGGTA 639 QRGSLAR 844 RDGSLTS1049 RSDHLTT 1254 1000 599 TGGTTGGTA 640 QNSAMRK 845 RDGSLTS 1050 RSDHLTT 1255 1000
600 GAGTCGGAA 641 QSANLAR 846 RSDELRT 1051 RSDNLAR 1256 206.7
601 GAGTCGGAA 642 RSANLTR 847 RLDGLRT 1052 RSDNLAR 1257 606.7
602 GAGTCGGAA 643 RSANLTR 848 RQDTLVG 1053 RSDNLAR 1258 616.7
603 GAGTCGGAA 644 QSGNLAR 849 RSDELRT 1054 RSDNLAR 1259 166.7 606 GGGGAGGATC 645 TTSNLRR 850 RSDNLQR 1055 RSDHLSR 1260 0.2
TABLE 3
SEQ SEQ SEQ SEQ Kd
^1^4 TARGET ID FI ID F2 ID F3 ID (nM)
897 GAGGAGGTGA 1261 RSDALAR 1347 RSDNLAR 1433 RSDNLVR 1519 0.07
828 GCGGAGGACC 1262 EKANLTR 1348 RSDNLAR 1434 RSDERKR 1520 0.1 884 GAGGAGGTGA 1263 RSDSLTR 1349 RSDNLAR 1435 RSDNLVR 1521 0.15 817 GAGGAGGTGA 1264 RSDSLTR 1350 RSDNLAR 1436 RSDNLAR 1522 0.31 666 GCGGAGGCGC 1265 RSDDLTR 1351 RSDNLTR 1437 RSDTLKK 1523 0.5
829 GCGGAGGACC 1266 EKANLTR 1352 RSDNLAR 1438 RSDTLKK 1524 ■ 0.52 670 GACGTGGAGG 1267 RSDNLAR 1353 RSDALAR 1439 DRSNLTR 1525 0.57 801 AAGGAGTCGC 1268 RSADLRT1354 RSDNLAR 1440 RSDNLTQ 1526 0.85 668 GTGGAGGCCA 1269 ERGTLAR 1355 RSDNLAR 1441 RSDALAR 1527 1.13 895 ATGGATTCAG 1270 QSHDLTK 1356 TSGNLVR 1442 RSDALTQ 1528 1.4 799 GGGGGAGCTG 1271 QSSDLQR 1357 QRAHLER 1443 RSDHLSR 1529 1.85 798 GGGGGAGCTG 1272 QSSDLQR 1358 QSGHLQR 1444 RSDHLSR 1530 3 842 GAGGTGGGCT 1273 DRSHLTR 1359 RSDALAR 1445 RSDNLAR 1531 5.4 894 TCAGTGGTAT 1274 QRSALAR 1360 RSDALSR 1446 QSHDLTK 1532 6.15 892 ATGGATTCAG 1275 QSHDLTK 1361 QQSNLVR 1447 RSDALTQ 1533 6.2 888 TCAGTGGTAT 1276 QSSSLVR 1362 RSDALSR 1448 QSHDLTK 1534 14 739 GCGGGCGGGC 1277 RSDHLTR 1363 ERGHLTR 1449 RSDDLRR 1535 16.5 850 CAGGCTGTGG 1278 RSDALTR 1364 QSSDLTR 1450 RSDNLRE1536 17 797 GCAGAGGCTG 1279 QSSDLQR 1365 RSDNLAR 1451 QSGDLTR 1537 17.5 891 TCAGTGGTAT 1280 QSSSLVR 1366 RSDALSR 1452 QSGSLRT1538 18.5 887 TCAGTGGTAT 1281 QRSALAR 1367 RSDALSR 1453 QSGDLRT1539 23.75
672 TCGGACGTGG 1282 RSDALAR 1368 DRSNLTR 1454 RSDELRT 1540 24 836 GGGGAGGCCC 1283 ERGTLAR 1369 RSDNLAR 1455 RSDHLSR 1541 24.25 674 GCGGCGTCGG 1284 RSDELRT 1370 RADTLRR 1456 RSDTLKK 1542 27.5 849 GGGGCCCTGG 1285 RSDALRE 1371 DRSSLTR 1457 RSDHLTQ 1543 29.05 825 GAATGGGCAG 1286 QSGSLTR 1372 RSDHLTT 1458 QSGNLTR 1544 37.3
673 GCGGGTGTCT 1287 DRSALAR 1373 QSSHLAR 1459 RSDTLKK 1545 48.33 848 GGGGAGGCCC 1288 DRSSLTR 1374 RSDNLAR 1460 RSDHLSR 1546 49.5 662 AGAGCGGCAC 1289 QTGSLTR1375 RSDELQR 1461 QSGHLNQ 1547 50 667 GAGTCGGACG 1290 DRSNLTR 1376 RSDELRT 1462 RSDNLAR 1548 50
803 GCAGCGGCTC 1291 QSSDLQR 1377 RSDELQR 1463 QSGSLTR 1549 57.5 671 TCGGACGAGT 1292 RSDNLAR 1378 DRSNLTR 1464 RSDELRT 1550 64 851 GAGATGGATC 1293 QSSNLQR 1379 RRDVLMN 1465 RLHNLQR 1551 74
804 GCAGCGGCTC 1294 QSSDLQR 1380 RSDDLNR 1466 QSGSLTR 1552 82.5 669 GACGAGTCGG 1295 RSDELRT 1381 RSDNLAR 1467 DRSNLTR 1553 90
682 GCTGCAGGAG 1296 RSDHLAR 1382 QSGDLTR 1468 QSSDLSR 1554 90 845 GAGATGGATC 1297 QSSNLQR 1383 RSDALRQ 1469 RLHNLQR 1555 112.5
663 AGAGCGGCAC 1298 QTGSLTR1384 RSDELQR 1470 KNWKLQA 1556 115 738 GCGGGGTCCG 1299 ERGTLTT1385 RSDHLSR 1471 RSDDLRR 1557 120
664 AGAGCGGCAC 1300 QTGSLTR1386 RADTLRR 1472 ASSRLAT 1558 125
833 GACTAGGACC 1301 EKANLTR 1387 RSDNLTK 1473 DRSNLTR 1559 136 685 GCTGCAGGAG 1302 RSDHLAR 1388 QSGSLTR 1474 QSSDLSR 1560 150 835 TAGGGAGCGT 1303 RADTLRR 1389 QSGHLTR 1475 RSDNLTT 1561 150 847 TAGGGAGCGT 1304 RSDDLTR 1390 QSGHLTR 1476 RSDNLTT 1562 150 818 GAATGGGCAG 1305 QSGSLTR 1391 RSDHLTT 1477 QSSNLVR 1563 167
834 GACTAGGACC 1306 EKANLTR 1392 RSDHLTT 1478 DRSNLTR 1564 186
837 GGGGCCCTGG 1307 RSDALRE 1393 DRSSLTR 1479 RSDHLSR 1565 222 764 GCAGAGGCTG 1308 TSGELVR1394 RSDNLAR 1480 QSGDLTR 1566 255
774 GCAGCGGTAG 1309 QRSALAR 1395 RSDELQR 1481 QSGDLTR 1567 258 765 GCCGAGGCCG 1310 ERGTLAR 1396 RSDNLAR 1482 ERGTLAR 1568 262.5
766 GCCGAGGCCG 1311 ERGTLAR 1397 RSDNLAR 1483 DRSDLTR 1569 262.5
775 GCAGCGGTAG 1312 QSGALTR 1398 RSDELQR 1484 QSGDLTR 1570 265 763 GCAGAGGCTG 1313 TSGELVR1399 RSDNLAR 1485 QSGSLTR 1571 275
838 GGGGCCCTGG 1314 RSDALRE 1400 DRSSLTR 1486 RSDHLTA 1572 300 841 GAGTGTGAGG 1315 RSDNLAR 1401 QSSHLAS 1487 RSDNLAR 1573 300 770 TTGGCAGCCT 1316 DRSSLTR 1402 QSGSLTR 1488 RSDSLTK 1574 325
767 GGGGGAGCTG 1317 QSSDLAR 1403 QSGHLQR 1489 RSDHLSR 1575 335 800 TTGGCAGCCT 1318 ERGTLAR 1404 QSGSLTR 1490 RSDSLTK 1576 400 832 GACTAGGACC 1319 EKANLTR 1405 RSDNLTT 1491 DRSNLTR 1577 408 844 GAGATGGATC 1320 QSSNLQR 1406 RSDALRQ 1492 RSDNLQR 1578 444
683 GCTGCAGGAG 1321 QSGHLAR 1407 QSGSLTR 1493 QSSDLSR 1579 500
805 GCAGCGGTAG 1322 QRSALAR 1408 RSDELQR 1494 QSGSLTR 1580 500
839 GAGTGTGAGG 1323 RSDNLAR 1409 TSDHLAS 1495 RSDNLAR 1581 625 840 GAGTGTGAGG 1324 RSDNLAR 1410 MSHHLKT 1496 RSDNLAR 1582 625
830 GGAGAGTCGG 1325 RSDELRT 1411 RSDNLAR 1497 QRAHLAR 1583 683
831 GGAGAGTCGG 1326 RSDDLTK 1412 RSDNLAR 1498 QRAHLAR 1584 700 684 GCTGCAGGAG 1327 RSAHLAR 1413 QSGSLTR 1499 QSSDLSR 1585 850 846 GAGATGGATC 1328 QSSNLQR 1414 RRDVLMN 1500 RSDNLQR 1586 889.5
819 AAGTAGGGTG 1329 QSSHLTR 1415 RSDNLTT 1501 RSDNLTQ 1587 1000
820 ACGGTAGTTA 1330 QSSALTR 1416 QRSALAR 1502 RSDTLTQ 1588 1000
821 ACGGTAGTTA 1331 NRATLAR 1417 QRSALAR 1503 RSDTLTQ 1589 1000
822 GTGTGCTGGT 1332 RSDHLTT 1418 ERQHLAT 1504 RSDALAR 1590 1000
823 GTGTGCTGGT 1333 RSDHLTK 1419 ERQHLAT 1505 RSDALAR 1591 1000
824 GTGTGCTGGT 1334 RSDHLTT 1420 DRSHLRT 1506 RSDALAR 1592 1000
885 GTGTGCTGGT 1335 RSDHLTK 1421 DRSHLRT 1507 RSDALAR 1593 1000
886 TCAGTGGTAT 1336 QSSSLVR 1422 RSDALSR 1508 QSGDLRT 1594 1000
889 ATGGATTCAG 1337 QSGSLTT 1423 QQSNLVR 1509 RSDALTQ 1595 1000
890 CTGGTATGTC 1338 QRSHLTT 1424 QRSALAR 1510 RSDALRE 1596 1000 896 AAGTAGGGTG 1339 TSGHLVR 1425 RSDNLTT 1511 RSDNLTQ 1597 1000
898 ACGGTAGTTA 1340 NRATLAR 1426 QSSSLVR 1512 RSDTLTQ 1598 1000
899 CTGGTATGTC 1341 QRSHLTT 1427 QSSSLVR 1513 RSDALRE 1599 1000
900 CTGGTATGTC 1342 MSHHLKE 1428 QSSSLVR 1514 RSDALRE 1600 1000
901 CTGGTATGTC 1343 MSHHLKE 1429 QRSALAR 1515 RSDALRE 1601 1000 773 GCAGCGGTAG 1344 QSGALTR 1430 RSDELQR 1516 QSGSLTR 1602 1250 768 GGGGGAGCTG 1345 QSSDLAR 1431 QRAHLER 1517 RSDHLSR 1603 2000 681 GCTGCAGGAG 1346 RSAHLAR 1432 QSGDLTR 1518 QSSDLSR 1604 3000
TABLE 4
SEQ FI SEQ. F2 SEQ. F3 SEQ Kd
SBS* TARGET IP ID ID ID (nM)
607 AAGGTGGCAG 1605 QSGDLTR 1707 RSDSLAR 1809 RLDNRTA 1911 6.5
608 TTGGCTGGGC 1606 GSWHLTR 1708 QSSDLQR 1810 RSDSLTK 1912 8
611 GTGGCTGCAG 1607 QSGDLTR 1709 QSSDLQR 1811 RSDALAR 1913 11.5
612 GTGGCTGCAG 1608 QSGTLTR 1710 QSSDLQR 1812 RSDALAR 1914 0.38
613 TTGGCTGGGC 1609 RSDHLAR 1711 QSSDLQR 1813 RGDALTS 1915 1.45
614 TTGGCTGGGC 1610 RSDHLAR 1712 QSSDLQR 1814 RSDSLTK 1916 2
616 GAGGAGGATG 1611 QSSNLQR 1713 RSDNLAR 1815 RSDNLQR 1917 0.08
617 AAGGGGGGG 1612 RSDHLSR 1714 RSDHLTR 1816 RKDNMTA 1918 1
618 AAGGGGGGG 1613 RSDHLSR 1715 RSDHLTR 1817 RKDNMTQ 1919 0.55
619 AAGGGGGGG 1614 RSDHLSR 1716 RSDHLTR 1818 RKDNMTN 1920 1.34
620 AAGGGGGGG 1615 RSDHLSR 1717 RSDHLTR 1819 RLDNRTA 1921 0.54
621 AAGGGGGGG 1616 RSDHLSR 1718 RSDHLTR 1820 RLDNRTQ 1922 0.75 624 ACGGATGTCT 1617 DRSALAR 1719 TSANLAR 1821 RSDTLRS 1923 7
628 TTGTAGGGGA 1618 RSDHLTR 1720 RSDNLTT 1822 RGDALTS 1924 130
629 TTGTAGGGGA 1619 RSSHLTR 1721 RSDNLTT 1823 RGDALTS 1925 150
630 CGGGGAGAGT 1620 RSDNLAR 1722 QSGHLQR 1824 RSDHLRE 1926 37.5
646 TTGGTGGAAG 1621 QSGNLAR 1723 RSDALAR 1825 RGDALTS 1927 35
647 TTGGTGGAAG 1622 QSANLAR 17*24 RSDALAR 1826 RGDALTS 1928 40
651 GTTGTGGAAT 1623 QSGNLSR 1725 RSDALAR 1827 NRATLAR 1929 67.5
652 TAGGAGGCTG 1624 QSSDLQR 1726 RSDNLAR 1828 RSDNLTT 1930 1.5
653 TAGGAGGCTG 1625 TTSDLTR 1727 RSDNLAR 1829 RSDNLTT 1931 5.5
654 TAGGCATAAA 1626 QSGNLRT 1728 QSGSLTR 1830 RSDNLTT 1932 105
655 TAGGCATAAA 1627 QSGNLRT 1729 QSSTLRR 1831 RSDNLTT 1933 1000
656 TAGGCATAAA 1628 QSGNLRT 1730 QSGSLTR 1832 RSDNLTS 1934 540
657 TAGGCATAAA 1629 QSGNLRT 1731 QSSTLRR 1833 RSDNLTS 1935 300
660 GAGGGAGTTC 1630 NRATLAR 1732 QSGHLTR 1834 RSDNLAR 1936 8.25
661 GAGGGAGTTC 1631 TTSALTR 1733 QSGHLTR 1835 RSDNLAR 1937 1.73 665 GCGGAGGCGC 1632 RSDDVTR 1734 RSDNLTR 1836 RSDDLRR 1938 12.5 689 AAGGCGGAGA 1633 RSDNLTR 1735 RSDELQR 1837 RLDNRTA 1939 82.5 692 AAGGCGGAGA 1634 RSDNLTR 1736 RSDELQR 1838 RSDNLTQ 1940 51
693 AAGGCGGAGA 1635 RSDNLTR 1737 RADTLRR 1839 RLDNRTA 1941 95
694 AAGGCGGAGA 1636 RSDNLTR 1738 RADTLRR 1840 RSDNLTQ 1942 28.5
695 GGGGGCGAGC 1637 RSSNLTR 1739 DRSHLAR 1841 RSDHLTR 1943 850
697 TGAGCGGCGG 1638 RSDELTR 1740 RSDELSR 1842 QSGHLTK 1944 200
698 TGAGCGGCGG 1639 RSDELTR 1741 RSDELSR 1843 QSHGLTS 1945 300
699 GCGGCGGCAG 1640 QSGSLTR 1742 RSDDLQR 1844 RSDERKR 1946 21.5
700 GCGGCGGCAG 1641 QSGDLTR 1743 RSDDLQR 1845 RSDERKR 1947 45
701 GCAGCGGAGC 1642 RSDNLAR 1744 RSDELQR 1846 QSGSLTR 1948 50.5
702 GCAGCGGAGC 1643 RSDNLAR 1745 RSDELQR 1847 QSGDLTR 1949 73.5
704 AAGGTGGCAG 1644 QSGDLTR 1746 RSDSLAR 1848 RSDNLTQ 1950 5
705 GGGGTGGGGC 1645 RSDHLAR 1747 RSDSLAR 1849 RSDHLSR 1951 0.01
706 GGGGTGGGGC 1646 RSDHLAR 1748 RSDSLLR 1850 RSDHLSR 1952 0.05
708 GAGTCGGAA 1647 QSANLAR 1749 RQDTLVG 1851 RSDNLAR 1953 300
709 GAGTCGGAA 1648 QSANLAR 1750 RKDVLVS 1852 RSDNLAR 1954 400
710 GAGTCGGAA 1649 QSGNLAR 1751 RLDGLRT 1853 RSDNLAR 1955 400
711 GAGTCGGAA 1650 QSGNLAR 1752 RQDTLVG 1854 RSDNLAR 1956 400
712 GGTGAGGAGT 1651 RSDNLAR 1753 RSDNLAR 1855 MSDHLSR 1957 9.5
713 GGTGAGGAGT 1652 RSDNLAR 1754 RSDNLAR 1856 MSHHLSR 1958 0.15
714 TGGGTCGCGG 1653 RSDELRR 1755 DRSALAR 1857 RSDHLTT 1959 200
715 TGGGTCGCGG 1654 RADTLRR 1756 DRSALAR 1858 RSDHLTT 1960 0.46
716 TTGGGAGCAC 1655 QSGSLTR 1757 QSGHLQR 1859 RGDALTS 1961 200
717 TTGGGAGCAC 1656 QSGSLTR 1758 QSGHLQR 1860 RSDALTK 1962 150
718 TTGGGAGCAC 1657 QSGSLTR 1759 QSGHLQR 1861 RSDALTR 1963 107.5
719 GGCATGGTGG 1658 RSDALTR 1760 RSDALTS 1862 DRSHLAR 1964 20 720 GAAGAGGATG 1659 TTSNLAR 1761 RSDNLAR 1863 QSGNLTR 1965 1.6 722 ATGGGGGTGG 1660 RSDALTR 1762 RSDHLTR 1864 RSDALRQ 1966 0.7
724 GGCATGGTGG 1661 RSDALTR 1763 RSDALRQ 1865 DRSHLAR 1967 2.5
725 GCTTGAGTTA 1662 QSSALAR 1764 QSGHLQK 1866 QSSDLQR 1968 3000
726 GAAGAGGATG 1663 QSSNLAR 1765 RSDNLAR 1867 QSGNLTR 1969 1.5
727 GCGGTGGCTC 1664 QSSDLTR 1766 RSDALSR 1868 RSDTLKK 1970 0.1
728 GGTGAGGAGT 1665 RSDNLAR 1767 RSDNLAR 1869 DSSKLSR 1971 15
729 GGAGGGGAGT 1666 RSDNLAR 1768 RSDHLSR 1870 QSGHLAR 1972 1000
730 TGGGTCGCGG 1667 RSDDLTR 1769 DRSALAR 1871 RSDHLTT 1973 1000 731 GTGGGGGAGA 1668 RSDNLAR 1770 RSDHLSR 1872 RSDALAR 1974 12
732 GCGGGTGGGG 1669 RSDHLAR 1771 QSSHLAR 1873 RSDDLTR 1975 22.5
733 GCGGGTGGGG 1670 RSDHLAR 1772 QSSHLAR 1874 RSDTLKK 1976 0.32
734 GGGGCTGGGT 1671 RSDHLAR 1773 QSSDLSR 1875 RSDHLSR 1977 0.25
735 GCGGTGGCTC 1672 QSSDLTR 1774 RSDALSR 1876 RSDERKR 1978 0.05 736 GAGGTGGGGA 1673 RSDHLAR 1775 RSDALSR 1877 RSDNLSR 1979 0.47 737 GGAGGGGAGT 1674 RSDNLAR 1776 RSDHLSR 1878 QRGHLSR 1980 1000
740 AAGGTGGCAG 1675 QSGSLTR 1777 RSDALAR 1879 RSDNRTA 1981 12.5
741 AAGGCTGAGA 1676 RSDNLTR 1778 QSSDLQR 1880 RSDNLTQ 1982 15
742 ACGGGGTTAT 1677 QRGALAS 1779 RSDHLSR 1881 RSDTLKQ 1983 29
743 ACGGGGTTAT 1678 QRGALAS 1780 RSDHLSR 1882 RSDTLTQ 1984 10
744 ACGGGGTTAT 1679 QRSALAS 1781 RSDHLSR 1883 RSDTLKQ 1985 8.33
745 ACGGGGTTAT 1680 QRSALAS 1782 RSDHLSR 1884 RSDTLTQ 1986 12.5
746 CTGGAAGCAT 1681 QSGSLTR 1783 QSGNLAR 1885 RSDALRE 1987 2.07
747 CTATTTTGGG 1682 RSDHLTT 17*84 QSSALRT 1886 QSGALRE 1988 2000
748 TTGGACGGCG 1683 DSGHLTR 1785 DRSNLER 1887 RGDALTS 1989 112.3
749 TTGGACGGCG 1684 DRSHLTR 1786 DSSNLTR 1888 RGDALTS 1990 11.33 750 GAGGGAGCGA 1685 RSDELTR 1787 QSAHLAR 1889 RSDNLAR 1991 52
751 GGTGAGGAGT 1686 RSDNLAR 1788 RSDNLAR 1890 NRSHLAR 1992 7
752 GAGGTGGGGA 1687 RSHHLAR 1789 RSDALSR 1891 RSDNLSR 1993' 31
757 CGGGCGGCTG 1688 QSSDLRR 1790 RSDELQR 1892 RSDHLRE 1994 14.5
758 CGGGCGGCTG 1689 QSSDLRR 1791 RADTLRR 1893 RSDHLRE 1995 16.5
759 TTGGACGGCG 1690 DSGHLTR 1792 DSSNLTR 1894 RGDALTS 1996 37
760 TTGGACGGCG 1691 DRSHLTR 1793 DRSNLER 1895 RGDALTS 1997 148.5
761 GCGGTGGCTC 1692 QSSDLQR 1794 RSDALSR 1896 RSDERKR 1998 6
762 GCGGTGGCTC 1693 QSSDLQR 1795 RSDALSR 1897 RSDTLKK 1999 18
776 ATGGACGGGT 1694 RSDHLAR 1796 DRSNLER 1898 RSDSLNQ 2000 0.4
777 ATGGACGGGT 1695 RSDHLAR 1797 DRSNLTR 1899 RSDALSA2001 3.4
779 CGGGGAGCAG 1696 QSGSLTR 1798 QSGHLTR 1900 RSDHLAE2002 0.5
780 CGGGGAGCAG 1697 QSGSLTR 1799 QSGHLTR 1901 RSDHLRA2003 0.5 781 GGGGAGCAGC 1698 RSSNLRE 1800 RSDNLAR 1902 RSDHLTR 2004 4.25 783 TTGGGAGCGG 1699 RSDELTR 1801 QSGHLQR 1903 RGDALTS 2005 2000
785 TTGGGAGCGG 1700 RSDTLKK 1802 QSGHLQR 1904 RSDALTS 2006 50
786 TTGGGAGCGG 1701 RSDTLKK 1803 QSGHLQR 1905 RGDALRS 2007 2000 787 AGGGAGGATG 1702 QSDNLAR 1804 RSDNLAR 1906 RSDHLTQ 2008 4
826 GAGGGAGCGA 1703 RSDELTR 1805 QSGHLAR 1907 RSDNLAR 2009 2.75
827 GAGGGAGCGA 1704 RADTLRR 1806 QSGHLAR 1908 RSDNLAR 2010 1.2
882 GCGTGGGCGT 1705 RSDELTR 1807 RSDHLTT 1909 RSDERKR 2011 0.01
883 GCGTGGGCGT 1706 RSDELTR 1808 RSDHLTT 1910 RSDERKR 2012 1
TABLE 5
SEQ SEQ SEQ SEQ Kd
SBS# TARGET ID (nM)
ID Ei F2 ID F3 ID
903 ATGGAAGGG 2013 RSDHLAR 2513 QSGNLAR 3013 RSDALRQ 3513 1.027 904 AAGGGTGAC 2014 DSSNLTR 2514 QSSHLAR 3014 RSDNLTQ 3514 1 905 GTGGTGGTG 2015 RSSALTR 2515 RSDSLAR 3015 RSDSLAR 3515 1.15 908 AAGGTCTCA 2016 QSGDLRT 2516 DRSALAR 3016 RSDNLRQ 3516 50 909 GTGGAAGAA 2017 QSGNLSR 2517 QSGNLQR 3017 RSDALAR 3517 16.4 910 ATGGAAGAT 2018 QSSNLAR 2518 QSGNLQR 3018 RSDALAQ 3518 0.03 911 ATGGGTGCA 2019 QSGSLTR 2519 QSSHLAR 3019 RSDALAQ 3519 0.91 912 TCAGAGGTG 2020 RSDSLAR 2520 RSDNLTR 3020 QSGDLRT 3520 0.135 914 CAGGAAAAG 2021 RSDNLTQ 2521 QSGNLAR 3021 RSDNLRE 3521 1.26 915 CAGGAAAAG 2022 RSDNLRQ 2522 QSGNLAR 3022 RSDNLRE 3522 45.15 916 GAGGAAGGA 2023 QSGHLAR 2523 QSGNLAR 3023 RSDNLQR 3523 1.3 919 TCATAGTAG 2024 RSDNLTT 2524 RSDNLRT 3024 QSGDLRT 3524 250 920 GATGTGGTA 2025 QSSSLVR 2525 RSDSLAR 3025 TSANLSR 3525 4 921 AAGGTCTCA 2026 QSGDLRT 2526 DPGALVR 3026 RSDNLRQ 3526 11 922 AAGGTCTCA 2027 QSHDLTK 2527 DRSALAR 3027 RSDNLRQ 3527 4 923 AAGGTCTCA 2028 QSHDLTK 2528 DPGALVR 3028 RSDNLRQ 3528 2 926 GTGGTGGTG 2029 RSDALTR 2529 RSDSLAR 3029 RSDSLAR 3529 7.502 927 CAGGTTGAG 2030 RSDNLAR 2530 TSGSLTR 3030 RSDNLRE 3530 3.61 928 CAGGTTGAG 2031 RSDNLAR 2531 QSSALTR 3031 RSDNLRE 3531 25 929 CAGGTAGAT 2032 QSSNLAR 2532 QSATLAR 3032 RSDNLRE 3532 1.3 931 GAGGAAGAG 2033 RSDNLAR 2533 QSSNLVR 3033 RSDNLAR 3533 2 932 ATGGAAGGG 2034 RSDHLAR 2534 QSSNLVR 3034 RSDALRQ 3534 797 933 GACGAGGAA 2035 QSANLAR 2535 RSDNLAR 3035 DRSNLTR 3535 500 934 ATGGAAGAT 2036 QSSNLAR 2536 QSGNLQR 3036 RSDALTS 3536 0.07 935 ATGGGTGCA 2037 QSGSLTR 2537 QSSHLAR 3037 RSDALTS 3537 0.91 937 GTGGGGGCT 2038 QSSDLTR 2538 RSDHLTR 3038 RSDSLAR 3538 0.03 938 GTGGGGGCT 2039 QSSDLRR 2539 RSDHLTR 3039 RSDSLAR 3539 0.049 939 GGGGGCTGG 2040 RSDHLTT 2540 DRSHLAR 3040 RSDHLSK 3540 0.352 940 GGGGGCTGG 2041 RSDHLTK 2541 DRSHLAR 3041 RSDHLSK 3541 1.5 941 GGGGCTGGG 2042 RSDHLAR 2542 QSSDLRR 3042 RSDKLSR 3542 0.077 942 GGGGCTGGG 2043 RSDHLAR 2543 QSSDLRR 3043 RSDHLSK 3543 0.13 943 GGGGCTGGG 2044 RSDHLAR 2544 TSGELVR 3044 RSDKLSR 3544 0.067 944 GGGGCTGGG 2045 RSDHLAR 2545 TSGELVR 3045 RSDHLSK 3545 027 945 GGTGCGGTG 2046 RSDSLTR 2546 RADTLRR 3046 MSHHLSR 3546 027 946 GGTGCGGTG 2047 RSDSLTR 2547 RSDVLQR 3047 MSHHLSR 3547 027 947 GGTGCGGTG 2048 RSDSLTR 2548 RSDELQR 3048 QSSHLAR 3548 013 948 GGTGCGGTG 2049 RSDSLTR 2549 RSDVLQR 3049 QSSHLAR 3549 017 962 GAGGCGGCA 2050 QSGSLTR 2550 RSDELQR 3050 RSDNLAR 3550 015 963 GAGGCGGCA 2051 QSGSLTR 2551 RSDDLQR 3051 RSDNLAR 3551 015 964 GCGGCGGTG 2052 RSDALAR 2552 RSDELQR 3052 RSDERKR 3552 0.041 965 GCGGCGGCC 2053 ERGDLTR 2553 RSDELQR 3053 RSDERKR 3553 3.1 966 GAGGAGGCC 2054 ERGTLAR 2554 RSDNLSR 3054 RSDNLAR 3554 0.028 967 GAGGAGGCC 2055 DRSSLTR 2555 RSDNLSR 3055 RSDNLAR 3555 0.055 968 GAGGCCGCA 2056 QSGSLTR 2556 DRSSLTR 3056 RSDNLAR 3556 1.4 969 GAGGCCGCA 2057 QSGSLTR 2557 DRSDLTR 3057 RSDNLAR 3557 0.275
970 GTGGGCGCC 2058 ERGTLAR 2558 DRSHLAR 3058 RSDALAR 3558 1.859
971 GTGGGCGCC 2059 DRSSLTR 2559 DRSHLAR 3059 RSDALAR 3559 0.144
972 GTGGGCGCC 2060 ERGDLTR 2560 DRSHLAR 3060 RSDALAR 3560 1.748
973 GCCGCGGTC 2061 DRΞALTR 2561 RSDELQR 3061 ERGTLAR 3561 0.6
974 GCCGCGGTC 2062 DRSALTR 2562 RSDELQR 3062 DRSDLTR 3562 0.038
975 CAGGCCGCT 2063 QSSDLTR 2563 DRSSLTR 3063 RSDNLRE 3563 1.1
976 CAGGCCGCT 2064 QSSDLTR 2564 DRSDLTR 3064 RSDNLRE 3564 4.12
977 CTGGCAGTG 2065 RSDSLTR 2565 QSGSLTR 3065 RSDALRE 3565 0.017
978 CTGGCAGTG 2066 RSDSLTR 2566 QSGDLTR 3066 RSDALRE 3566 1.576
979 CTGGCGGCG 2067 RSSDLTR 2567 RSDELQR 3067 RSDALRE 3567 1.59
980 CTGGCGGCG 2068 RSDDLTR 2568 RSDELQR 3068 RSDALRE 3568 2.2
981 CAGGCGGCG 2069 RSDDLTR 2569 RSDELQR 3069 RSDNLRE 3569 0.375
982 CCGGGCTGG 2070 RSDHLTT 2570 DRSHLAR 3070 RSDELRE 3570 0.03
983 CCGGGCTGG 2071 RSDHLTK 2571 DRSHLAR 3071 RSDELRE 3571 1.385
984 GACGGCGAG 2072 RSDNLAR 2572 DRSHLAR 3072 DRSNLTR 3572 1.6
985 GACGGCGAG 2073 RSDNLAR 2573 DRSHLAR 3073 EKANLTR 3573 0.965
986 GGTGCTGAT 2074 QSSNLQR 2574 QSSDLQR 3074 MSHHLSR 3574 1.6
987 GGTGCTGAT 2075 QSSNLQR 2575 QSSDLQR 3075 TSGHLVR 3575 33.55
988 GGTGCTGAT 2076 TSGNLVR 2576 QSSDLQR 3076 MSHHLSR 3576 0.15
989 GGTGAGGGG 2077 RSDHLAR 2577 RSDNLAR 3077 MSHHLSR 3577 1.9
990 AAGGTGGGC 2078 DRSHLTR 2578 RSDSLAR 3078 RSDNLTQ 3578 5.35
991 AAGGTGGGC 2079 DRSHLTR 2579 SSGSLVR 3079 RSDNLTQ 3579 0.06
993 GGGGCTGGG 2080 RSDHLAR 2580 TSGELVR 3080 RSDHLSR 3580 3.1
994 GGGGGCTGG 2081 RSDHLTK 2581 DRSHLAR 3081 RSDHLSR 3581 0.03
995 GGGGAGGAA 2082 QSANLAR 2582 RSDNLAR 3082 RSDHLSK 3582 0.08
996 CAGTTGGTC 2083 DRSALAR 2583 RSDALTS 3083 RSDNLRE 3583 9.6
997 AGAGAGGCT 2084 QSSDLTR 2584 RSDNLAR 3084 QSGHLNQ 3584 1.65
998 ACGTAGTAG 2085 RSANLRT 2585 RSDNLTK 3085 RSDTLKQ 3585 0.23
999 AGAGAGGCT 2086 QSSDLTR 2586 RSDNLAR 3086 QSGKLTQ 3586 0.6
1000 CAGTTGGTC 2087 DRSALAR 2587 RSDALTR 3087 RSDNLRE 3587 11.15
1001 GGAGCTGAC 2088 EKANLTR 2588 QSSDLSR 3088 QRAHLAR 3588 1.8
1002 GCGGAGGAG 2089 RSDNLVR 2589 RSDNLAR 3089 RSDERKR 3589 0.028
1003 ACGTAGTAG 2090 RSANLRT 2590 RSDNLTK 3090 RSDTLRS 3590 0.118
1004 ACGTAGTAG 2091 RSDNLTT 2591 RSDNLTK 3091 RSDTLRS 3591 1.4
1006 GTAGGGGCG 2092 RSDDLTR 2592 RSDHLTR 3092 QRASLTR 3592 0.898
1007 GAGAGAGAT 2093 QSSNLQR 2593 QSGHLTR 3093 RLHNLAR 3593 167
1008 GAGATGGAG 2094 RSDNLSR 2594 RSDSLTQ 3094 RLHNLAR 3594 0.4
1009 GAGATGGAG 2095 RSDNLSR 2595 RSDSLTQ 3095 RSDNLSR 3595 1.9
1010 GAGAGAGAT 2096 QSSNLQR 2596 QSGHLTR 3096 RSDNLAR 3596 8.2
1011 TTGGTGGCG 2097 RSADLTR 2597 RSDSLAR 3097 RSDSLTK 3597 0.03
1012 GACGTAGGG 2098 RSDHLTR 2598 QSSSLVR 3098 DRSNLTR 3598 0.032
1013 GAGAGAGAT 2099 QSSNLQR 2599 QSGHLNQ 3099 RSDNLAR 3599 0.15
1014 GACGTAGGG 2100 RSDHLTR 2600 QSGSLTR 3100 DRSNLTR 3600 0.01
1015 GCGGAGGAG 2101 RSDNLVR 2601 RSDNLAR 3101 RSDTLKK 3601 0.008
1016 CAGTTGGTC 2102 DRSALAR 2602 RSDSLTK 3102 RSDNLRE 3602 0.09
1017 CTGGATGAC 2103 EKANLTR 2603 TSGNLVR 3103 RSDALRE 3603 0.233
1018 GTAGTAGAA 2104 QSANLAR 2604 QSSSLVR 3104 QRASLAR 3604 7.2
1019 AGGGAGGAG 2105 RSDNLAR 2605 RSDNLAR 3105 RSDHLTQ 3605 0.022
1020 ACGTAGTAG 2106 RSDNLTT 2606 RSDNLTK 3106 RSDTLKQ 3606 0.69
1022 GAGGAGGTG 2107 RSDALAR 2607 RSDNLAR 3107 RSDNLAR 3607 0.01 1024 GGGGAGGAA 2108 QSANLAR 2608 RSDNLAR 3108 RSDHLSR 3608 0.08 1025 GAGGAGGTG 2109 QSSALTR2609 QSSSLVR 3109 RSDTLTQ 3609 0.115 1026 GTGGCTTGT 2110 MSHHLKE 2610 QSSDLSR 3110 RSDALAR 3610 0.076 1027 GCGGCGGTG 2111 RSDALAR 2611 RSDELQR 3111 RSDELQR 3611 0.054 1032 GGTGCTGAT 2112 TSGNLVR 2612 QSSDLQR 3112 TSGHLVR 3612 0.52 1033 GTGTTCGTG 2113 RSDALAR 2613 DRSALTT 3113 RSDALAR 3613 685.2 1034 GTGTTCGTG 2114 RSDALAR 2614 DRSALTK3114 RSDALAR 3614 14.55 1035 GTGTTCGTG 2115 RSDALAR 2615 DRSALRT 3115 RSDALAR 3615 56 1037 GTAGGGGCA 2116 QSGSLTR 2616 RSDHLSR 3116 QRASLAR 3616 0.05 1038 GTAGGGGCA 2117 QTGELRR 2617 RSDHLSR 3117 QRASLAR 3617 0.152 1039 GGGGCTGGG 2118 RSDHLSR 2618 TSGELVR 3118 RSDHLTR 3618 1.37 1040 GGGGCTGGG 2119 RSDHLSR 2619 QSSDLQR 3119 RSDHLSK 3619 0.05 1041 TCATAGTAG 2120 RSDNLTT 2620 RSDNLRT 3120 QSHDLTK 3620 2.06. 1043 CAGGGAGAG 2121 RSDNLAR 2621 QSGHLTR 3121 RSDNLRE 3621 0.16 1044 CAGGGAGAG 2122 RSDNLAR 2622 QRAHLER 3122 RSDNLRE 3622 1.07 1045 GGGGCAGGA 2123 QSGHLAR 2623 QSGSLTR 3123 RSDHLSR 3623 0.15 1046 GGGGCAGGA 2124 QSGHLAR 2624 QSGDLRR 3124 RSDHLSR 3624 0.09 1047 GGGGCAGGA 2125 QRAHLER2625 QSGSLTR 3125 RSDHLSR 3625 24.7 1048 CAGGCTGTA 2126 QSGALTR 2626 QSSDLQR 3126 RSDNLRE 3626 1.387 1049 CAGGCTGTA 2127 QRASLAR 2627 QSSDLQR 3127 RSDNLRE 3627 55.6 1050 CAGGCTGTA 2128 QSSSLVR 2628 QSSDLQR 3128 RSDNLRE 3628 0.125 1051 GAGGCTGAG 2129 RSDNLTR 2629 QSSDLQR 3129 RSDNLVR 3629 0.02 1052 TAGGACGGG 2130 RSDHLAR 2630 EKANLTR 3130 RSDNLTT 3630 0.28 1053 TAGGACGGG 2131 RSDHLAR 2631 DRSNLTR 3131 RSDNLTT 3631 0.025 1054 GCTGCAGGG 2132 RSDHLAR 2632 QSGSLTR 3132 QSSDLQR 3632 0.033 1055 GCTGCAGGG 2133 RSDHLAR 2633 QSGSLTR 3133 TSGDLTR3633 18.73 1056 GCTGCAGGG 2134 RSDHLAR 2634 QSGSLTR 3134 QSSDLQR 3634 0.045 1057 GCTGCAGGG 2135 RSDHLAR 2635 QSGDLTR 3135 TSGDLTR3635 0.483 1058 GGGGCCGCG 2136 RSDELTR 2636 DRSSLTR 3136 RSDHLSR 3636 6.277 1059 GGGGCCGCG 2137 RSDELTR 2637 DRSDLTR 3137 RSDHLSR 3637 0.152 1060 GCGGAGGCC 2138 ERGTLAR 2638 RSDNLAR 3138 RSDERKR 3638 0.69 1061 GTTGCGGGG 2139 RSDHLAR 2639 RSDELQR 3139 QSSALTR3639 0.165 1062 GTTGCGGGG 2140 RSDHLAR 2640 RSDELQR 3140 TSGSLTR3640 0.068 1063 GTTGCGGGG 2141 RSDHLAR 2641 RSDELQR 3141 MSHALSR3641 0.96 1064 GCGGCAGTG 2142 RSDALTR 2642 QSGSLTR 3142 RSDERKR 3642 0.453 1065 TGGGGCGGG 2143 RSDHLA 2643 DRSHLAR 3143 RSDHLTT 3643 1.37 1066 GAGGGCGGT 2144 QSSHLTR 2644 DRSHLAR 3144 RSDNLVR 3644 0.15 1067 GAGGGCGGT 2145 TSGHLVR 2645 DRSHLAR 3145 RSDNLVR 3645 1.37 1068 GCAGGGGGC 2146 DRSHLTR 2646 RSDHLTR 3146 QSGDLTR 3646 2.05 1069 GCAGGCGGT 2147 DRSHLTR 2647 RSDHLTR 3147 QSGSLTR 3647 0.1 1070 GGGGCAGGC 2148 DRSHLTR 2648 QSGSLTR 3148 RSDHLSR 3648 0.456 1071 GGGGCAGGC 2149 DRSHLTR 2649 QSGDLTR 3149 RSDHLSR 3649 0.2 1072 GGATTGGCT 2150 QSSDLTR 2650 RSDALTT 3150 QRAHLAR 3650 0.46 1073 GGATTGGCT 2151 QSSDLTR 2651 RSDALTK3151 QRAHLAR 3651 1.37 1075 GTGTTGGCG 2152 RSDELTR 2652 RSDALTK3152 RSDALTR 3652 0.915 1076 GCGGCAGCG 2153 RSDELTR 2653 QSGSLTR 3153 RSDERKR 3653 4.1 1077 GCGGCAGCG 2154 RSDELTR 2654 QSGDLRR 3154 RSDERKR 3654 6.2 1078 GGGGGGGCC 2155 ERGTLAR 2655 RSDHLSR 3155 RSDHLSR 3655 0.2 1079 GGGGGGGCC 2156 ERGDLTR 2656 RSDHLSR 3156 RSDHLSR 3656 4.1 1080 CTGGAGGCG 2157 RSDELTR 2657 RSDNLAR 3157 RSDALRE 3657 1.37 1081 GGGGAGGTG 2158 RSDALTR 2658 RSDNLTR 3158 RSDHLSR 3658 0.05 1082 CTGGCGGCG 2159 RSDELTR 2659 RSDELTR 3159 RSDALRE 3659 0.152 1083 CTGGTGGCA 2160 QSGDLTR 2660 RSDALSR 3160 RSDALRE 3660 0.152 1084 GGTGAGGCG 2161 RSDELTR 2661 RSDNLAR 3161 MSHHLSR 3661 0.5 1085 GGTGAGGCG 2162 RSDELTR 2662 RSDNLAR 3162 QSSHLAR 3662 0.46 1086 GGGGCTGGG 2163 RSDHLSR 2663 QSSDLQR 3163 RSDHLTR 3663 0.1 1087 CGGGCGGCC 2164 ERGDLTR 2664 RSDELQR 3164 RSDHLAE 3664 1.24 1088 CGGGCGGCC 2165 ERGDLTR 2665 RSDELQR 3165 RSDHLRE 3665 0.905 1089 GACGAGGCT 2166 QSSDLRR 2666 RSDNLAR 3166 DRSNLTR 3666 0.171 1090 AAGGCGCTG 2167 RSDALRE 2667 RSDELQR 3167 RSDNLTQ 3667 30.3 1091 GTAGAGGAC 2168 DRSNLTR 2668 RSDNLAR 3168 QRASLAR 3668 0.085 1092 GCCTTGGCT 2169 QSSDLRR 2669 RGDALTS 3169 DRSDLTR 3669 2.735 1093 GCGGAGTCG 2170 RSADLRT2670 RSDNLAR 3170 RSDERKR 3670 0.046 1094 GCGGTTGGT 2171 TSGHLVR 2671 QSSALTR3171 RSDERKR 3671 12.34 1095 GGGGGAGCC 2172 ERGDLTR 2672 QRAHLER 3172 RSDHLSR 3672 0.395 1096 GGGGGAGCC 2173 DRSSLTR 2673 QRAHLER 3173 RSDHLSR 3673 0.019 1097 GAGGCCGAA 2174 QSANLAR 2674 DCRDLAR 3174 RSDNLAR 3674 0.77 1098 GCCGGGGAG 2175 RSDNLTR 2675 RSDHLTR 3175 DRSDLTR 3675 0.055 1099 GCGGAGTCG 2176 TSGHLVR 2676 TSGSLTR3176 RSDERKR 3676 0.45 1100 GTGTTGGTA 2177 QSGALTR 2677 RGDALTS 3177 RSDALTR 3677 1.4 1101 ATGGGAGTT 2178 TTSALTR2678 QRAHLER 3178 RSDALRQ 3678 0.065 1102 AAGGCAGAA 2179 QSANLAR 2679 QSGSLTR 3179 RSDNLTQ 3679 8.15 1103 AAGGCAGAA 2180 QSANLAR 2680 QSGDLTR 3180 RSDNLTQ 3680 1.4 1104 CGGGCAGCT 2181 QSSDLRR 2681 QSGSLTR 3181 RSDHLRE 3681 0.08 1105 CTGGCAGCC 2182 ERGDLTR 2682 QSGDLTR 3182 RSDALRE 3682 2.45 1106 CTGGCAGCC 2183 DRSSLTR 2683 QSGDLTR 3183 RSDALRE 3683 0.19 1107 GCGGGAGTT 2184 QSSALAR 2684 QRAHLER 3184 RSDERKR 3684 0.06 1108 CAGGCTGGA 2185 QSGHLAR 2685 TSGELVR 3185 RSDNLRE 3685 0.007 1109 AGGGGAGCC 2186 ERGDLTR 2686 QRAHLER 3186 RSDHLTQ 3686 347 1110 AGGGGAGCC 2187 DRSSLTR 2687 QRAHLER 3187 RSDHLTQ 3687 095 1111 CTGGTAGGG 2188 RSDHLAR 2688 QSSSLVR 3188 RSDALRE 3688 095 1112 CTGGTAGGG 2189 RSDHLAR 2689 QSATLAR 3189 RSDALRE 3689 125 1113 CTGGGGGCA 2190 QSGDLTR 2690 RSDHLTR 3190 RSDALRE 3690 0.06 1114 CAGGTTGAT 2191 QSSNLAR 2691 TSGSLTR3191 RSDNLRE 3691 2.75 1115 CAGGTTGAT 2192 QSSNLAR 2692 QSSALTR3192 RSDNLRE 3692 0.7 1116 CCGGAAGCG 2193 RSDELTR 2693 QSSNLVR 3193 RSDELRE 3693 12.3 1117 GCAGCGCAG 2194 RSSNLRE2694 RSDELTR 3194 QSGSLTR 3694 2.85 1118 TAGGGAGTC 2195 DRSALTR 2695 QRAHLER 3195 RSDNLTT 3695 1.4 1119 TGGGAGGGT 2196 TSGHLVR 2696 RSDNLAR 3196 RSDHLTT 3696 0.1 1120 AGGGACGCG 2197 RSDELTR 2697 DRSNLTR 3197 RSDHLTQ 3697 2.735 1121 CTGGTGGCC 2198 ERGDLTR 2698 RSDALTR 3198 RSDALRE 3698 2.76 1122 CTGGTGGCC 2199 DRSSLTR 2699 RSDALTR 3199 RSDALRE 3699 0.101 1123 TAGGAAGCA 2200 QSGSLTR 2700 QSGNLAR 3200 RSDNLTT 3700 0.065 1124 GTGGATGGA 2201 QSGHLAR 2701 TSGNLVR 3201 RSDALTR 3701 0.101 1126 TTGGCTATG 2202 RSDALTS 2702 TSGELVR 3202 RGDALTS 3702 0.46 1127 CAGGGGGTT 2203 QSSALAR 2703 RSDHLTR 3203 RSDNLRE 3703 0.1 1128 AAGGTCGCC 2204 ERGDLTR 2704 DPGALVR 3204 RSDNLTQ 3704 5.45 1130 GGTGCAGAC 2205 DRSNLTR 2705 QSGDLTR 3205 MSHHLSR 3705 0.1 1131 GTGGGAGCC 2206 ERGDLTR 2706 QRAHLER 3206 RSDALTR 3706 0.95 1132 GGGGCTGGA 2207 QSGHLAR 2707 TSGELVR 3207 RSDHLSR 3707 0.055 1133 GGGGCTGGA 2208 QRAHLER 2708 TSGELVR 3208 RSDHLSR 3708 0.5 1134 TGGGGGTGG 2209 RSDHLTT 2709 RSDHLTR 3209 RSDHLTT 3709 0.067 1135 GCGGCGGGG 2210 RSDHLAR 2710 RSDELQR 3210 RSDERKR 3710 .025 1136 CCGGGAGTG 2211 RSDALTR 2711 QRAHLER 3211 RSDTLRE 3711 .225 1137 CCGGGAGTG 2212 RSSALTR2712 QRAHLER 3212 RSDTLRE 3712 .085 1138 CAGGGGGTA 2213 QSGALTR 2713 RSDHLTR 3213 RSDNLRE 3713 ,027 1139 ACGGCCGAG 2214 RSDNLAR 2714 DRSDLTR 3214 RSDTLTQ 3714 535 1140 AAGGGTGCG 2215 RSDELTR 2715 QSSHLAR 3215 RSDNLTQ 3715 0.3 1141 ATGGACTTG 2216 RGDALTS 2716 DRSNLTR 3216 RSDALTQ 3716 1.7 1148 TTGGAGGAG 2217 RSDNLTR 2717 RSDNLTR 3217 RGDALTS 3717 0.006 1149 TTGGAGGAG 2218 RSDNLTR 2718 RSDNLTR 3218 RSDALTK3718 0.004 1150 GAAGAGGCA 2219 QSGSLTR 2719 RSDNLTR 3219 QSGNLTR 3719 0.004 1151 GTAGTATGG 2220 RSDHLTT 2720 QRSALAR 3220 QRASLAR 3720 1.63 1152 AAGGCTGGA 2221 QSGHLAR 2721 TSGELVR 3221 RSDNLTQ 3721 1.605 1153 AAGGCTGGA 2222 QRAHLAR 2722 TSGELVR 3222 RSDNLTQ 3722 8.2 1154 CTGGCGTAG 2223 RSDNLTT 2723 RSDELQR 3223 RSDALRE 3723 1.04 1156 ATGGTTGAA 2224 QSANLAR 2724 QSSALTR3224 RSDALRQ 3724 7.2 1157 ATGGTTGAA 2225 QSANLAR 2725 TSGSLTR3225 RSDALRQ 3725 0.885 1158 AGGGGAGAA 2226 QSANLAR 2726 QSGHLTR 3226 RSDHLTQ 3726 0 . 1 1159 AGGGGAGAA 2227 QSANLAR 2727 QRAHLER 3227 RSDHLTQ 3727 0 . 555 1160 TGGGAAGGC 2228 DRSHLAR 2728 QSSNLVR 3228 RSDHLTT 3728 0 . 415 1161 GAGGCCGGC 2229 DRSHLAR 2729 DRSDLTR 3229 RSDNLAR 3729 0 . 45 1162 GTGTTGGTA 2230 QSGALTR 2730 RADALMV3230 RSDALTR 3730 0 . 465 1163 GTGTGAGCC 2231 ERGDLTR 2731 QSGHLTT3231 RSDALTR 3731 1 . 45 1164 GTGTGAGCC 2232 ERGDLTR 2732 QSVHLQS 3232 RSDALTR 3732 15 . 4 1165 GCGAAGGTG 2233 RSDALTR 2733 RSDNLTQ 3233 RSDERKR 3733 1 . 4 1166 GCGAAGGTG 2234 RSDALTR 2734 RSDNLTQ 3234 RSSDRKR3734 0 . 195 1167 GCGAAGGTG 2235 RSDALTR 2735 RSDNLTQ 3235 RSHDRKR 3735 0 . 95 1168 AAGGCGCTG 2236 RSDALRE 2736 RSSDLTR 3236 RSDNLTQ 3736 2 . 8 1169 GTAGAGGAC 2237 DRSNLTR 2737 RSDNLAR 3237 QSSSLVR 3737 0 . 053 1170 GCCTTGGCT 2238 QSSDLRR 2738 RADALMV3238 DRSDLTR 3738 2 . 75 1171 GCGGAGTCG 2239 RSDDLRT2739 RSDNLAR 3239 RSDERKR 3739 0 . 18 1172 GCCGGGGAG 2240 RSDNLTR 2740 RSDHLTR 3240 ERGDLTR 3740 0 . 01 1173 GCTGAAGGG 2241 RSDHLSR 2741 QSGNLAR 3241 QSSDLRR 3741 0 . 008 1174 GCTGAAGGG 2242 RSDHLSR 2742 QSSNLVR 3242 QSSDLRR 3742 0 . 018 1175 AAGGTCGCC 2243 DRSDLTR 2743 DPGALVR 3243 RSDNLTQ 3743 8 . 9 1176 GTGGGAGCC 2244 DRSDLTR 2744 QRAHLER 3244 RSDALTR 3744 4 . 1 1177 CCGGGCGCA 2245 QSGSLTR 2745 DRSHLAR 3245 RSDTLRE 3745 4 . 1 1178 GAGGATGGC 2246 DRSHLAR 2746 TSGNLVR 3246 RSDNLAR 3746 0 . 085 1179 GCAGCGCAG 2247 RSSNLRE2747 RSSDLTR 3247 QSGSLTR 3747 2 . 735 1180 AAGGAAAGA 2248 QSGHLNQ 2748 QSGNLAR 3248 RSDNLTQ 3748 4 . 825 1181 TTGGCTATG 2249 RSDALRQ 2749 TSGELVR 3249 RGDALTS 3749 8 . 2 1182 CAGGAAGGC 2250 DRSHLAR 2750 QSGNLAR 3250 RSDNLRE 3750 1 . 48 1183 CAGGAAGGC 2251 DRSHLAR 2751 QSSNLVR 3251 RSDNLRE 3751 1 . 935 1184 AAGGAAAGA 2252 KNWKLQA2752 QSGNLAR 3252 RSDNLTQ 3752 2 . 785 1185 AAGGAAAGA 2253 KNWKLQA2753 QSHNLAR3253 RSDNLTQ 3753 5 . 25 1186 GCCGAGGTG 2254 RSDSLLR 2754 RSKNLQR3254 ERGTLAR 3754 27 . 5 1187 CTGGTGGGC 2255 DRSHLAR 2755 RSDALTR 3255 RSDALRE 3755 0 . 006 1188 GTAGTATGG 2256 RSDHLTT 2756 QSSSLVR 3256 QRASLAR 3756 2 . 74 1189 ATGGTTGAA 2257 QSANLAR 2757 TSGALTR 3257 RSDALRQ 3757 1 . 51 1190 ATGGCAGTG 2258 RSDALTR 2758 QSGDLTR 3258 RSDSLNQ 3758 1 . 484 1191 ATGGCAGTG 2259 RSDALTR 2759 QSGSLTR 3259 RSDSLNQ 3759 5 . 325 1192 ATGGCAGTG 2260 RSDALTR 2760 QSGDLTR 3260 RSDALTQ 3760 2 . 364 1193 ATGGCAGTG 2261 RSDALTR 2761 QSGSLTR 3261 RSDALTQ 3761 3.125 1194 GAGAAGGTG 2262 RSDALTR 2762 RSDNRTA 3262 RSDNLTR 3762 2.19 1195 GAGAAGGTG 2263 RSDALTR 2763 RSDNRTA 3263 RSSNLTR 3763 2.8 1197 GAAGGTGCC 2264 ERGDLTR 2764 MSHHLSR 3264 QSGNLTR 3764 14.8 1199 ATGGAGAAG 2265 RSDNRTA 2765 RSDNLTR 3265 RSDALTQ 3765 3.428 1200 ATGGAGAAG 2266 RSDNRTA 2766 RSSNLTR 3266 RSDALTQ 3766 16.87 1201 ATGGAGAAG 2267 RSDNRTA 2767 RSHNLTR 3267 RSDALTQ 3767 14.8 1202 CTGGAGTAC 2268 DRSNLRT 2768 RSDNLTR 3268 RSDALRE 3768 2.834 1203 GGAGTACTG 2269 RSDALRE 2769 QRSALAR 3269 QRAHLAR 3769 2.945 1204 GGAGTACTG 2270 RSDALRE 2770 QSSSLVR 3270 QRAHLAR 3770 4.38 1205 CGGGCAGCT 2271 QSSDLRR 2771 QSGDLTR 3271 RSDHLRE 3771 0.9 1206 GCGGGAGTT 2272 TTSALTR 2772 QRAHLER 3272 RSDERKR 3772 0.034 1207 CAGGCTGGA 2273 QRAHLER 2773 TSGELVR 3273 RSDNLRE 3773 0.45 1209 CCGGAAGCG 2274 RSDELTR 2774 QSSNLVR 3274 RSDTLRE 3774 19.28 1211 GCAGCGCAG 2275 RSDNLRE 2775 RSDELTR 3275 QSGSLTR 3775 6.5 1212 CAGGGGGTT 2276 TTSALTR 2776 RSDHLTR 3276 RSDNLRE 3776 0.05 1213 GAAGAAGAG 2277 RSDNLTR 2777 QSSNLVR 3277 QSGNLTR 3777 12.3 1214 ATGGGAGTT 2278 TTSALTR 2778 QRAHLER 3278 RSDALTQ 3778 0.46 1215 GTGGGGGCT 2279 QSSDLRR 2779 RSDHLTR 3279 RSDALTR 3779 0.003 1217 GAAGAGGCA 2280 QSGSLTR 2780 RSDNLTR 3280 QSANLTR 3780 0.004 1218 GCGGTGAGG 2281 RSDHLTQ 2781 RSQALTR 3281 RSDERKR 3781 0.46 1219 AAGGAAAGG 2282 RSDHLTQ 2782 QSHNLAR 3282 RSDNLTQ 3782 0.68 1220 AAGGAAAGG 2283 RSDHLTQ 2783 QSGNLAR 3283 RSDNLTQ 3783 0.175 1221 AAGGAAAGG 2284 RSDHLTQ 2784 QSSNLVR 3284 RSDNLTQ 3784 1.4 1222 CAGGAGGGC 2285 DRSHLAR 2785 RSDNLAR 3285 RSDNLRE 3785 0.155 1223 ATGGACTTG 2286 RSDALTK 2786 DRSNLTR 3286 RSDALTQ 3786 7 1224 ATGGACTTG 2287 RADALMV 2787 DRSNLTR 3287 RSDALTQ 3787 12 1227 GAATAGGGG 2288 RSDHLSR 2788 RSDHLTK 3288 QSGNLAR 3788 25 1228 ACGGCCGAG 2289 RSDNLAR 2789 DRSDLTR 3289 RSDDLTQ 3789 12 1229 AAGGGTGCG 2290 RSDELTR 2790 MSHHLSR 3290 RSDNLTQ 3790 8.2 1230 AAGGGAGAC 2291 DRSNLTR 2791 QSGHLTR 3291 RSDNLTQ 3791 0.383 1231 AAGGGAGAC 2292 DRSNLTR 2792 QRAHLER 3292 RSDNLTQ 3792 0 213 1232 TGGGACCTG 2293 RSDALRE 2793 DRSNLTR 3293 RSDHLTT 3793 0 113 1233 TGGGACCTG 2294 RSDALRE 2794 DRSNLTR 3294 RSDHLTT 3794 0.635 1234 GAGTAGGCA 2295 QSGSLTR 2795 RSDNLTK 3295 RSDNLAR 3795 0.101 1236 GAGTAGGCA 2296 QSGSLTR 2796 RSDHLTT 3296 RSDNLAR 3796 0.065 1237 GAAGGAGAG 2297 RSDNLAR 2797 QRAHLER 3297 QSGNLAR 3797 0.065 1238 CTGGATGTT 2298 QSSALAR 2798 TSGNLVR 3298 RSDALRE 3798 0.313 1239 CAGGACGTG 2299 RSDALTR 2799 DPGNLVR 3299 RSDNLKD 3799 0.144 1240 GGGGAGGCA 2300 QSGSLTR 2800 RSDNLTR 3300 RSDHLSR 3800 0.056 1241 GAGGTGTCA 2301 QSHDLTK 2801 RSDALAR 3301 RSDNLAR 3801 0.027 1242 GGGGTTGAA 2302 QSANLAR 2802 TSGSLTR 3302 RSDHLSR 3802 0.02 1243 GGGGTTGAA 2303 QSANLAR 2803' QSSALTR 3303 RSDHLSR 3803 0.101 1244 GTCGCGGTG 2304 RSDALTR 2804 RSDELQR 3304 DRSALAR 3804 0.044 1245 GTCGCGGTG 2305 RSDALTR 2805 RSDELQR 3305 DSGSLTR 3805 0.102 1246 GTGGTTGCG 2306 RSDELTR 2806 TSGSLTR 3306 RSDALTR 3806 0.051 1247 GTGGTTGCG 2307 RSDELTR 2807 TSGALTR 3307 RSDALTR 3807 0.117 1248 GTCTAGGTA 2308 QSGALTR 2808 RSDNLTT 3308 DRSALAR 3808 5.14 1249 CCGGGAGCG 2309 RSDELTR 2809 QSGHLTR 3309 RSDTLRE 3809 0.26 1250 GAAGGAGAG 2310 RSDNLAR 2810 QSGHLTR 3310 QSGNLAR 3810 0.31 1252 CCGGCTGGA 2311 QRAHLER 2811 QSSDLTR 3311 RSDTLRE 3811 0.153 1253 CCGGGAGCG 2312 RSDELTR 2812 QRAHLER 3312 RSDTLRE 3812 0.228 1255 ACGTAGTAG 2313 RSDNLTT 2813 RSDNLTK 3313 RSDTLKQ 3813 0.69 1256 GGGGAGGAT 2314 QSSNLAR 2814 RSDNLQR 3314 RSDHLSR 3814 2 1257 GGGGAGGAT 2315 TTSNLAR2815 RSDNLQR 3315 RSDHLSR 3815 1 1258 GGGGAGGAT 2316 QSSNLRR2816 RSDNLQR 3316 RSDHLSR 3816 2 1259 GAGTGTGTG 2317 RSDSLLR 2817 DRDHLTR 3317 RSDNLAR 3817 1.5 1260 GAGTGTGTG 2318 RLDSLLR2818 DRDHLTR 3318 RSDNLAR 3818 1.8 1261 TGCGGGGCA 2319 QSGDLTR 2819 RSDHLTR 3319 RRDTLHR 3819 0.2 1262 TGCGGGGCA 2320 QSGDLTR 2820 RSDHLTR 3320 RLDTLGR 3820 3 1263 TGCGGGGCA 2321 QSGDLTR 2821 RSDHLTR 3321 DSGHLAS 3821 21 1264 AAGTTGGTT 2322 TTSALTR 2822 RADALMV 3322 RSDNLTQ 3822 0.21 1265 AAGTTGGTT 2323 TTSALTR 2823 RSDALTT3323 RSDNLTQ 3823 0.077 1266 CAGGGTGGC 2324 DRSHLTR 2824 QSSHLAR 3324 RSDNLRE 3824 6.1 1267 TAGGCAGTC 2325 DRSALTR 2825 QSGSLTR 3325 RSDNLTT 3825 6 1268 CTGTTGGCT 2326 QSSDLTR 2826 RADALMV 3326 RSDALRE 3826 1.52 1269 CTGTTGGCT 2327 QSSDLTR 2827 RSDALTT3327 RSDALRE 3827 12.3 1270 TTGGATGGA 2328 QSGHLAR 2828 TSGNLVR 3328 RSDALTK 3828 0.4 1271 GTGGCACTG 2329 RSDALRE 2829 QSGSLTR 3329 RSDALTR 3829 0.915 1272 CAGGAGTCC 2330 DRSSLTT2830 RSDNLAR 3330 RSDNLRE 3830 0.04 1273 CAGGAGTCC 2331 ERGDLTT2831 RSDNLAR 3331 RSDNLRE 3831 0.1 1274 GCATGGGAA 2332 QSANLSR 2832 RSDHLTT 3332 QSGSLTR 3832 0.306 1275 GCATGGGAA 2333 QRSNLVR 2833 RSDHLTT 3333 QSGSLTR 3833 326 1276 TAGGAAGAG 2334 RSDNLAR 2834 QRSNLVR 3334 RSDNLTT 3834 685 1277 GAAGAGGGG 2335 RSDHLAR 2835 RSDNLAR 3335 QSGNLTR 3835 421 1278 GAGTAGGCA 2336 QSGSLTR 2836 RSDNLRT3336 RSDNLAR 3836 019 1279 GAGGTGTCA 2337 QSGDLRT 2837 RSDALAR 3337 RSDNLAR 3837 0.025 1282 TCGGTCGCC 2338 ERGDLTR 2838 DPGALVR 3338 RSDELRT 3838 74.1 1287 GTGGTAGGA 2339 QSGHLAR 2839 QSGALAR 3339 RSDALTR 3839 0.152 1288 CAGGGTGGC 2340 DRSHLTR 2840 QSSHLAR 3340 RSDNLTE 3840 4.1 1289 TAGGCAGTC 2341 DRSALTR 2841 QSGSLTR 3341 RSDNLTK 3841 1.37 1290 GTGGTGATA 2342 QSGALTQ2842 RSHALTR3342 RSDALTR 3842 24.05 1291 GTGGTGATA 2343 QQASLNA2843 RSHALTR3343 RSDALTR 3843 20.55 1292 TTGGATGGA 2344 QSGHLAR 2844 TSGNLVR 3344 RSDALTT 3844 4.12 1293 AAGGTAGGT 2345 TSGHLVR 2845 QSGALAR 3345 RSDNLTQ 3845 0.457 1294 AAGGTAGGT 2346 MSHHLSR 2846 QSGALAR 3346 RSDNLTQ 3846 2.75 1295 CAGGAGTCC 2347 DRSSLTT2847 RSDNLAR 3347 RSDNLTE 3847 0.116 1296 CAGGAGTCC 2348 ERGDLTT2848 RSDNLAR 3348 RSDNLTE 3848 37 1297 TAGGAAGAG 2349 RSDNLAR 2849 QRSNLVR 3349 RSDNLTK 3849 0.05 1298 CAGGACGTG 2350 RSDLATR2850 DPGNLVR 3350 RSDNLTE 3850 0.05 1300 GTCTAGGTA 2351 QSGALTR 2851 RSDNLTK 3351 DRSALAR 3851 0.46 1302 CCGGCTGGA 2352 QSGHLTR 2852 QSSDLTR 3352 RSDTLRE 3852 0.05 1303 TAGGAGTTT 2353 QRSALAS 2853 RSDNLAR 3353 RSDNLTT 3853 0.088 1306 CTGGCCTTG 2354 RSDALTT2854 DCRDLAR 3354 RSDALRE 3854 2.285 1308 TGGGCAGCC 2355 ERGTLAR 2855 QSGSLTR 3355 RSDHLTT 3855 0.305 1309 TAGGAGTTT 2356 QSSALAS2856 RSDNLAR 3356 RSDNLTT 3856 0.184 1310 TAGGAGTTT 2357 TTSALAS2857 RSDNLAR 3357 RSDNLTT 3857 0.075 1311 TGGGCAGCC 2358 ERGDLAR2858 QSGSLTR 3358 RSDHLTT 3858 0.91 1312 GGGGCGTGA 2359 QSGHLTK2859 RSDELQR 3359 RSDHLSR 3859 0.23 1313 GGGGCGTGA 2360 QSGHLTT2860 RSDELQR 3360 RSDHLSR 3860 0.09 1314 GTACAGTAG 2361 RSDNLTT 2861 RSDNLRE 3361 QSSSLVR 3861 3.09 1315 GTACAGTAG 2362 RSDNLTT 2862 RSDNLTE 3362 QSSSLVR 3862 9.27 1318 ATGGTGTGT 2363 TSSHLAS 2863 RSDALAR 3363 RSDALAQ 3863 0.048
1319 ATGGTGTGT 2364 MSHHLTT 2864 RSDALAR 3364 RSDALAQ 3864 0.228 1320 TTGGGAGAG 2365 RSDNLAR 2865 QRAHLER 3365 RSDALTT 3865 0.044
1321 TTGGGAGAG 2366 RSDNLAR 2866 QRAHLER 3366 RADALMV 3866 0.127
1322 GTGGGAATA 2367 QSGALTQ2867 QSGHLTR 3367 RSDALTR 3867 0.799 1323 GTGGGAATA 2368 QLTGLNQ2868 QSGHLTR 3368 RSDALTR 3868 0.744 1324 GTGGGAATA 2369 QQASLNA2869 QSHHLTR3369 RSDALTR 3869 18.52 1325 TTGGTTGGT 2370 TSGHLVR 2870 TSGSLTR 3370 RSDALTK 3870 0.306 1326 TTGGTTGGT 2371 TSGHLVR2871 QSSALTR3371 RSDALTK 3871 4.385 1327 TTGGTTGGT 2372 TSGHLVR 2872 TSGSLTR 3372 RSDALTT 3872 0.566 1328 TTGGTTGGT 2373 TSGHLVR2873 QSSALTR3373 RSDALTT 3873 7.95
1329 CTGGCCTGG 2374 RSDHLTT 2874 DRSDLTR 3374 RSDALRE 3874 0.68
1330 GAGGTGTGA 2375 QSGHLTT 2875 RSDALTR 3375 RSDNLAR 3875 0.175 1331 CTGGCCTGG 2376 RSDHLTT 2876 DCRDLAR3376 RSDALRE 3876 0.388 1334 CCGGCGCTG 2377 RSDALRE 2877 RSSDLTR 3377 RSDDLRE 3877 0.31 1335 GACGCTGGC 2378 DRSHLTR 2878 QSSDLTR 3378 DSSNLTR 3878 1.4 1336 CGGGCTGGA 2379 QSGHLAR 2879 QSSDLTR 3379 RSDHLAE 3879 1.4 1337 CGGGCTGGA 2380 QSSHLAR2880 QSSDLTR3380 RSDHLAE 3880 0.235 1338 GGGATGGCG 2381 RSDELTR 2881 RSDALTQ 3381 RSDHLSR 3881 1.04
1339 GGGATGGCG 2382 RSDELTR 2882 RSDSLTQ 3382 RSDHLSR 3882 0.569
1340 GGGATGGCG 2383 RSDELTR 2883 RSDALTQ 3383 RSHHLSR3883 0.751
1341 GGGATGGCG 2384 RSDELTR 2884 RSDSLTQ 3384 RSHHLSR3884 4.1
1342 CAGGCGCAG 2385 RSDNLRE 2885 RSSDLTR 3385 RSDNLTE 3885 0.68
1343 CAGGCGCAG 2386 RSDNLTT 2886 RTSTLTR3386 RSDNLTE 3886 37.04
1344 CCGGGCGAC 2387 DRSNLTR 2887 DRSHLAR 3387 RSDTLRE 3887 2.28
1346 GATGTGTGA 2388 QSGHLTT 2888 RSDALAR 3388 TSANLSR 3888 0.153
1347 CAGTGAATG 2389 RSDALTS 2889 QSHHLTT 3389 RSDNLTE 3889 8.23
1348 GGGTCACTG 2390 RSDALT 2890 QAATLTT3390 RSDHLSR 3890 2.58 1350 CAGTGAATG 2391 RSDALTQ 2891 QSGHLTT 3391 RSDNLTE 3891 74.1 1351 GGGTCACTG 2392 RSDALRE 2892 QSHDLTK3392 RSDHLSR 3892 0.234 1352 GTGTGGGTC 2393 DRSALAR 2893 RSDHLTT 3393 RSDALTR 3893 0.023 1353 CTGGCGAGA 2394 QSGHLNQ 2894 RSDELQR 3394 RSDALRE 3894 56.53 1354 CTGGCGAGA 2395 KNWKLQA2895 RSDELQR 3395 RSDALRE 3895 20.85 1355 GCTTTGGCA 2396 QSGSLTR2896 RSDALTT3396 QSSDLTR 3896 0.172 1356 GCTTTGGCA 2397 QSGSLTR 2897 RADALMV 3397 QSSDLTR 3897 0.034 1357 GACTTGGTA 2398 QSSSLVR2898 RSDALTT3398 DRSNLTR 3898 0.032 1358 GACTTGGTA 2399 QSSSLVR 2899 RADALMV 3399 DRSNLTR 3899 0.05 1360 CAGTTGTGA 2400 QSGHLTT 2900 RADALMV 3400 RSDNLTE 3900 41.7 1361 AAGGAAAAA 2401 QKTNLDT2901 QSGNLQR 3401 RSDNLTQ 3901 0.835 1362 AAGGAAAAA 2402 QSGNLNQ2902 QSGNLQR3402 RSDNLTQ 3902 0.332 1363 AAGGAAAAA 2403 QKTNLDT2903 QRSNLVR 3403 RSDNLTQ 3903 74.1 1364 ATGGGTGAA 2404 QSANLSR 2904 QSSHLAR 3404 RSDALAQ 3904 1.22 1365 ATGGGTGAA 2405 QRSNLVR 2905 QSSHLAR 3405 RSDALAQ 3905 0.152 1366 ATGGGTGAA 2406 QSANLSR 2906 TSGHLVR 3406 RSDALAQ 3906 22.63 1367 ATGGGTGAA 2407 QRSNLVR 2907 TSGHLVR 3407 RSDALAQ 3907 1.028 1368 CTGGGAGAT 2408 QSSNLAR 2908 QRAHLER 3408 RSDALRE 3908 0.051 1369 CTGGGAGAT 2409 QSSNLAR 2909 QSGHLTR 3409 RSDALRE 3909 0.227 1373 GTGGTGGGC 2410 DRSHLTR 2910 RSDALSR 3410 RSDALTR 3910 0.025 1374 CCGGCGGTG 2411 RSDALTR 2911 RSDELQR 3411 RSDELRE 3911 0.003 1375 CCGGCGGTG 2412 RSDALTR 2912 RSDDLQR 3412 RSDELRE 3912 0.008 1376 CCGGCGGTG 2413 RSDALTR 2913 RSDERKR 3413 RSDELRE 3913 0.858 1377 CCGGCGGTG 2414 RSDALTR 2914 RSDELQR 3414 RSDDLRE3914 0.012
1378 CCGGCGGTG 2415 RSDALTR 2915 RSDDLQR 3415 RSDDLRE3915 0.012
1379 CCGGCGGTG 2416 RSDALTR2916 RSDERKR3416 RSDDLRE3916 0.25
1380 GCCGACGGT 2417 QSSHLTR 2917 DRSNLTR 3417 ERGDLTR 3917 0.076
1381 GCCGACGGT 2418 QSSHLTR2918 DPGNLVR3418 ERGDLTR3918 0.23
1382 GCCGACGGT 2419 QSSHLTR2919 DRSNLTR3419 DCRDLAR3919 3.1
1383 GCCGACGGT 2420 QSSHLTR2920 DPGNLVR3420 DCRDLAR3920 1.74
1384 GGTGTGGGC 2421 DRSHLTR2921 RSDALSR3421 MSHHLSR3921 0.013
1385 TGGGCAAGA 2422 QSGHLNQ2922 QSGSLTR 3422 RSDHLTT3922 0.229
1386 TGGGCAAGA 2423 ENWKLQA2923 QSGSLTR 3423 RSDHLTT3923 0.193
1389 CTGGCCTGG 2424 RSDHLTT 2924 DCRDLAR 3424 RSDALRE 3924 0.175
1393 TGGGAAGCT 2425 QSSDLRR2925 QSGNLAR3425 RSDHLTT3925 0.1
1394 TGGGAAGCT 2426 QSSDLRR2926 QSGNLAR3426 RSDHLTK3926 0.04
1395 GAAGAGGGA 2427 QSGHLQR2927 RSDNLAR3427 QSGNLAR3927 0.025
1396 GAAGAGGGA 2428 QRAHLAR2928 RSDNLAR3428 QSGNLAR3928 0.107
1397 GAAGAGGGA 2429 QSSHLAR2929 RSDNLAR3429 QSGNLAR3929 0.14
1398 TAATGGGGG 2430 RSDHLSR2930 RSDHLTT3430 QSGNLRT3930 0.065
1399 TGGGAGTGT 2431 TKQHLKT2931 RSDNLAR3431 RSDHLTT3931 0.1
1400 CCGGGTGAG 2432 RSDNLAR2932 QSSHLAR3432 RSDDLRE3932 0.371 1401 GAGTTGGCC 2433 ERGTLAR2933 RADALMV3433 RSDNLAR3933 0.167 1402 CTGGAGTTG 2434 RGDALTS 2934 RSDNLAR 3434 RSDALRE3934 0.15 1403 ATGGCAATG 2435 RSDALTQ2935 QSGSLTR3435 RSDALTQ3935 0.07 1404 GAGGCAGGG 2436 RSDHLSR2936 QSGSLTR3436 RSDNLAR3936 0.022 1405 GAGGCAGGG 2437 RSDHLSR2937 QSGDLTR3437 RSDNLAR3937 0.045 1406 GAAGCGGAG 2438 RSDNLAR2938 RSDELTR3438 QSGNLAR3938 0.025 1407 GCGGGCGCA 2439 QSGSLTR2939 DRSHLAR3439 RSDERKR3939 0.585 1408 CCGGCAGGG 2440 RSDHLSR2940 QSGSLTR3440 RSDELRE3940 0.305 1409 CCGGCAGGG 2441 RSDHLSR2941 QSGSLTR3441 RSDDLRE3941 0.153
1410 CCGGCGGCG 2442 RSDELTR2942 RSDELQR 3442 RSDELRE3942 0.814
1411 TGAGGCGAG 2443 RSDNLAR2943 DRSHLAR 3443 QSGHLTK3943 0.282
1412 CTGGCCGTG 2444 RSDSLLR2944 ERGTLAR 3444 RSDALRE3944 0.172
1413 CTGGCCGCG 2445 RSDELTR 2945 DRSDLTR 3445 RSDALRE 3945 0.152
1414 CTGGCCGCG 2446 RSDELTR2946 ERGTLAR3446 RSDALRE3946 0.914
1415 GCGGCCGAG 2447 RSDNLAR2947 DRSDLTR 3447 RSDELQR3947 0.102
1416 GCGGCCGAG 2448 RSDNLAR2948 ERGTLAR 3448 RSDELQR3948 0.153
1417 GAGTTGGCC 2449 ERGTLAR2949 RGDALTS 3449 RSDNLAR3949 1.397
1418 CTGGAGTTG 2450 RADALMV 2950 RSDNLAR 3450 RSDALRE 3950 0.241
1422 GGGTCGGCG 2451 RSDELTR 2951 RSDDLTT 3451 RSDHLSR 3951 0.064
1423 GGGTCGGCG 2452 RSDELTR2952 RSDDLTK3452 RSDHLSR3952 0.034
1424 CAGGGCCCG 2453 RSDELRE2953 DRSHLAR3453 RSDNLRE3953 1.37 1427 CAGGGCCCG 2454 RSDDLRE2954 DRSHLAR3454 RSDNLTE3954 0.271 1428 TGAGGCGAG 2455 RSDNLAR2955 DRSHLAR3455 QSVHLQS3955 0.102
1429 TGAGGCGAG 2456 RSDNLAR2956 DRSHLAR3456 QSGHLTT3956 0.074
1430 TCGGCCGCC 2457 ERGTLAR2957 DRSDLTR3457 RSDDLTK3957 0.352 1431 TCGGCCGCC 2458 ERGTLAR2958 DRSDLTR3458 RSDDLAS3958 6.17 1432 TCGGCCGCC 2459 ERGTLAR2959 ERGTLAR3459 RSDDLTK3959 1.778 1434 CTGGCCGTG 2460 RSDSLLR 2960 DRSDLTR 3460 RSDALRE 3960 0.051 1435 TAATGGGGG 2461 RSDHLSR2961 RSDHLTT 3461 QSGNLTK3961 0.057 1436 TGGGAGTGT 2462 TSDHLAS2962 RSDNLAR3462 RSDHLTT3962 0.026
1439 GGAGTGTTA 2463 QRSALAS2963 RSDALAR3463 QSGHLQR3963 0.075
1440 GGAGTGTTA 2464 QSGALTK2964 RSDALAR3464 QSGHLQR3964 0.035 1441 ATAGCTGGG 2465 RSDHLSR 2965 QSSDLTR 3465 QSGALTQ3965 0.262 1442 TGCTGGGCC 2466 ERGTLAR 2966 RSDHLTT 3466 DRSHLTK3966 0.36 1443 TGGAAGGAA 2467 QSGNLAR 2967 RSDNLTQ 3467 RSHHLTT3967 0.22 1444 TGGAAGGAA 2468 QSGNLAR 2968 RSDNLTQ 3468 RSSHLTT3968 0.09 1445 TGGAAGGAA 2469 QSGNLAR 2969 RLDNLTA3469 RSHHLTT3969 0.182 1446 TGGAAGGAA 2470 QSGNLAR 2970 RLDNLTA3470 RSSHLTT3970 0.42 1454 GGAGAGGCT 2471 QSSDLRR 2971 RSDNLAR 3471 QSGHLQR 3971 0.01 1455 CGGGATGAA 2472 QSANLSR 2972 TSGNLVR 3472 RSDHLRE 3972 0.043 1456 GGAGAGGCT 2473 QSSDLRR 2973 RSDNLAR 3473 QRAHLAR 3973 0.016 1457 GCAGAGGAA 2474 QSANLSR 2974 RSDNLAR 3474 QSGSLTR 3974 0.014 1460 TTGGGGGAG 2475 RSDNLAR 2975 RSDHLTR 3475 RADALMV 3975 0.007 1461 GACGAGGAG 2476 RSANLAR2976 RSDNLTR 3476 DRSNLTR 3976 0.014 1462 CGGGATGAA 2477 QSGNLAR 2977 TSGNLVR 3477 RSDHLRE 3977 0.05 1463 GAGGCTGTT 2478 TTSALTR 2978 QSSDLTR 3478 RSDNLAR 3978 0.003 1464 GACGAGGAG 2479 RSDNLAR 2979 RSDNLTR 3479 DRSNLTR 3979 0.002 1465 CTGGGAGTT 2480 TTSALTR 2980 QSGHLQR 3480 RSDALRE 3980 0.018 1466 CTGGGAGTT 2481 NRATLAR 2981 QSGHLQR 3481 RSDALRE 3981 0.017 1468 GGTGATGTC 2482 DRSALTR 2982 TSGNLVR 3482 MSHHLSR 3982 0.08 1469 GGTGATGTC 2483 DRSALTR 2983 TSGNLVR 3483 TSGHLVR 3983 0.28 1470 GGTGATGTC 2484 DRSALTR 2984 TSGNLVR 3484 QRAHLER 3984 0.156 1471 CTGGTTGGG 2485 RSDHLSR 2985 QSSALTR3485 RSDALRE 3985 0.09 1472 TTGAAGGTT 2486 TTSALTR 2986 RSDNLTQ 3486 RADALMV 3986 3.22 1473 TTGAAGGTT 2487 TTSALTR 2987 RSDNLTQ 3487 RSDSLTT3987 0.47 1474 TTGAAGGTT 2488 QSSALAR 2988 RSDNLTQ 3488 RADALMV 3988 1.39 1475 TTGAAGGTT 2489 QSSALAR 2989 RSDNLTQ 3489 RLHSLTT3989 0.39 1476 TTGAAGGTT 2490 QSSALAR 2990 RSDNLTQ 3490 RSDSLTT3990 0.305 1477 GCAGCCCGG 2491 RSDHLRE 2991 DRSDLTR 3491 QSGSLTR 3991 2.31 1479 GAAAGTTCA 2492 QSHDLTK 2992 MSHHLTQ 3492 QSGNLAR 3992 37.04 1480 GAAAGTTCA 2493 NKTDLGK2993 TSGHLVQ 3493 QSGNLAR 3993 62.5 1481 GAAAGTTCA 2494 NKTDLGK2994 TSDHLAS 3494 RSDELRE 3994 37.04 1482 CCGTGTGAC 2495 DRSNLTR 2995 TSDHLAS 3495 RSDELRE 3995 111.1 1483 CCGTGTGAC 2496 DRSNLTR 2996 MSHHLTT 3496 RSDELRE 3996 20.8 1484 GAAGTGGTA 2497 QSSSLVR 2997 RSDALSR 3497 QSGNLAR 3997 0.01 1485 AAGTGAGCT 2498 QSSDLRR 2998 QSGHLTT 3498 RSDNLTQ 3998 1.537 1486 GGGTTTGAC 2499 DRSNLTR 2999 TTSALAS 3499 RSDHLSR 3999 0.085 1487 TTGAAGGTT 2500 TTSALTR 3000 RSDNLTQ 3500 RLHSLTT4000 0.188 1488 AAGTGGTAG 2501 QSSDLRR 3001 QSGHLTT 3501 RLDNRTQ4001 5.64 1490 CTGGTTGGG 2502 RSDHLSR 3002 TSGSLTR 3502 RSDALRE 4002 0.04 1491 AAGGGTTCA 2503 NKTDLGK3003 DSSKLSR3503 RLDNRTA 4003 4.12 1492 AAGTGGTAG 2504 RSDNLTT 3004 RSDHLTT 3504 RSDNLTQ 4004 1.37 1493 AAGTGGTAG 2505 RSDNLTT 3005 RSDHLTT 3505 RLDNRTQ4005 15.09 1494 GGGTTTGAC 2506 DRSNLTR 3006 QRSALAS 3506 RSDHLSR 4006 0.255 1496 TTGGGGGAG 2507 RSDNLAR 3007 RSDHLTR 3507 RSDALTT 4007 0.065 1497 GAGGCTCTT 2508 QSSALAR 3008 QSSDLTR 3508 RSDNLAR 4008 0.007 1498 GAGGTTGAT 2509 QSSNLAR 3009 QSSALTR3509 RSDNLAR 4009 0.101 1499 GAGGTTGAT 2510 QSSNLAR 3010 TSGALTR3510 RSDNLAR 4010 0.02 1500 GCAGAGGAA 2511 QSGNLAR 3011 RSDNLAR 3511 QSGSLTR 4011 0.003 1522 GCAATGGGT 2512 TSGHLVR 3012 RSDALTQ 3512 QSGDLTR 4012 0.08

Claims (92)

CLAIMS What is claimed is:
1. A zinc finger protein that binds to a target site, said zinc finger protein comprising a first (FI), a second (F2), and a third (F3) zinc finger, ordered FI, F2, F3 from N-terminus to C-terminus, said target site comprising, in 3' to 5' direction, a first (SI), a second (S2), and a third (S3) target subsite, each target subsite having the nucleotide sequence GNN, wherein if SI comprises GAA, FI comprises the amino acid sequence QRSNLNR; if S2 comprises GAA, F2 comprises the amino acid sequence QSGΝLAR; if S3 comprises GAA, F3 comprises the amino acid sequence QSGΝLAR; if SI comprises GAG, FI comprises the amino acid sequence RSDΝLAR; if S2 comprises GAG, F2 comprises the amino acid sequence RSDΝLAR; if S3 comprises GAG, F3 ■ comprises the amino acid sequence RSDNLTR; if SI comprises GAC, FI 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 SI comprises GAT, FI 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 SI comprises GGA, FI 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 SI comprises GGG, FI 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 SI comprises GGC, FI comprises the amino acid sequence DRSHLRT; if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR; if SI comprises GGT, FI 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 SI comprises GCA, FI comprises the amino acid sequence QSGSLTR; if S2 comprises GCA, F2 comprises QSGDLTR; if S3 comprises GCA, F3 comprises QSGDLTR; if SI comprises GCG, FI 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 SI comprises GCC, FI 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 SI comprises GCT, FI 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 SI comprises GTA, FI comprises the amino acid sequence QSGALTR; if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR; if SI comprises GTG, FI 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 SI comprises GTC, FI 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 SI comprises GAA and FI comprises the amino acid sequence QRSNLNR.
3. The zinc finger protein of claim 1 , wherein S2 comprises GAA and F2 comprises the amino acid sequence QSGΝLAR.
4. The zinc finger protein of claim 1, wherein S3 comprises GAA and F3 comprises the amino acid sequence QSGΝLAR.
5. The zinc finger protein of claim 1, wherein SI comprises GAG and FI comprises the amino acid sequence RSDΝLAR.
6. The zinc finger protein of claim 1, wherein S2 comprises GAG and F2 comprises the amino acid sequence RSDΝLAR.
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 SI comprises GAC and FI 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 SI comprises GAT and FI 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 TSGNLNR.
13. The zinc finger protein of claim 1, wherein S3 comprises GAT and F3 comprises the amino acid sequence TSAΝLSR.
14. The zinc finger protein of claim 1, wherein SI comprises GGA and FI 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 SI comprises GGG and FI 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 SI comprises GGC and FI 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 SI comprises GGT and FI 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 SI comprises GCA and FI 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 SI comprises GCG and FI 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 SI comprises GCC and FI 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 SI comprises GCT and FI 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 SI comprises GTA and FI 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 SI comprises GTG and FI 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 SI comprises GTC and FI 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 any of claims 1 to 46.
48. A polynucleotide encoding a polypeptide according to claim 47.
49. A method for designing a zinc finger protein comprising a first (FI), a second (F2), and a third (F3) zinc finger, ordered FI, F2, F3 from N-terminus to C-terminus that binds to a target site comprising, in 3' to 5' direction, a first (SI), a second (S2), and a third (S3) target subsite, each target subsite having the nucleotide sequence GNN, the method comprising the steps of selecting the FI zinc finger such that it binds to the SI target subsite, wherein if SI comprises GAA, FI comprises the amino acid sequence QRSNLNR; if SI comprises GAG, FI comprises the amino acid sequence RSDΝLAR; if SI comprises GAC, FI comprises the amino acid sequence DRSΝLTR; if SI comprises GAT, FI comprises the amino acid sequence QSSΝLAR; if SI comprises GGA, FI comprises the amino acid sequence QSGHLAR; if SI comprises GGG, FI comprises the amino acid sequence RSDHLAR; if SI comprises GGC, FI comprises the amino acid sequence DRSHLRT; if SI comprises GGT, FI comprises the amino acid sequence QSSHLTR; if SI comprises GCA, FI comprises QSGSLTR; if SI comprises GCG, FI comprises RSDDLTR; if S2 comprises GCG, F2 comprises RSDDLQR; if SI comprises GCC, FI comprises ERGTLAR; if SI comprises GCT, FI comprises the amino acid sequence QSSDLTR; if SI comprises GTA, FI comprises the amino acid sequence QSGALTR; if SI comprises GTG, FI comprises the amino acid sequence RSDALTR; if SI comprises GTC, FI comprises the amino acid sequence DRSALAR; selecting the F2 zinc finger such that it binds to the S2 target subsite, wherein S2 comprises GAA, F2 comprises the amino acid sequence QSGNLAR; if S2 comprises GAG, F2 comprises the amino acid sequence RSDNLAR; if S2 comprises GAC, F2 comprises the amino acid sequence DRSNLTR; if S2 comprises GAT, F2 comprises the amino acid sequence TSGNLNR; if S2 comprises GGA, F2 comprises the amino acid sequence QSGHLQR; if S2 comprises GGG, F2 comprises the amino acid sequence RSDHLSR; if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR; if S2 comprises GGT, F2 comprises the amino acid sequence TSGHLSR; if S2 comprises GCA, F2 comprises the amino acid sequence QSGDLTR; if S2 comprises GCC, F2 comprises the amino acid sequence DRSDLTR; if S2 comprises GCT, F2 comprises the amino acid sequence QSSDLTR; if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR; if S2 comprises GTG, F2 comprises the amino acid sequence RSDALSR; if S2 comprises GTC, F2 comprises the amino acid sequence DRSALAR; and selecting the F3 zinc finger such that it binds to the S3 target subsite, wherein if S3 comprises GAA, F3 comprises the amino acid sequence QSGΝLAR; if S3 comprises GAG, F3 comprises the amino acid sequence RSDNLTR; if S3 comprises GAC, F3 comprises the amino acid sequence DRSNLTR; if S3 comprises GAT, F3 comprises the amino acid sequence TSANLSR; if S3 comprises GGA, F3 comprises the amino acid sequence
QSGHLQR; if S3 comprises GGG, F3 comprises RSDHLSR; if S3 comprises GGT, F3 comprises the amino acid sequence TSGHLNR; if S3 comprises GCA, F3 comprises the amino acid sequence QSGDLTR; if S3 comprises GCG, F3 comprises the amino acid sequence RSDDLTR; if S3 comprises GCC, F3 comprises the amino acid sequence DRSDLTR; if S3 comprises GCT, F3 comprises the amino acid sequence QSSDLQR; if S3 comprises GTG, F3 comprises RSDALTR; and if S3 comprises GTC, F3 comprises the amino acid sequence DRSALAR; thereby designing a zinc finger protein that binds to a target site.
50. The method of claim 49, wherein SI comprises GAA and FI comprises the amino acid sequence QRSNLNR.
51. The method of claim 49, wherein S2 comprises GAA and F2 comprises the amino acid sequence QSGΝLAR.
52. The method of claim 49, wherein- S3 comprises GAA and F3 comprises the amino acid sequence QSGΝLAR.
53. The method of claim 49, wherein SI comprises GAG and FI comprises the amino acid sequence RSDΝLAR.
54. The method of claim 49, wherein S2 comprises GAG and F2 comprises the amino acid sequence RSDΝLAR.
55. The method of claim 49, wherein S3 comprises GAG and F3 comprises the amino acid sequence RSDNLTR.
56. The method of claim 49, wherein SI comprises GAC and FI comprises the amino acid sequence DRSNLTR.
57. The method of claim 49, wherein S2 comprises GAC and F2 comprises the amino acid sequence DRSNLTR.
58. The method of claim 49, wherein S3 comprises GAC and F3 comprises the amino acid sequence DRSNLTR.
59. The method of claim 49, wherein SI comprises GAT and FI comprises the amino acid sequence QSSNLAR.
60. The method of claim 49, wherein S2 comprises GAT and F2 comprises the amino acid sequence TSGNLVR.
61. The method of claim 49, wherein S3 comprises GAT and F3 comprises the amino acid sequence TSANLSR.
62. The method of claim 49, wherein SI comprises GGA and FI comprises the amino acid sequence QSGHLAR.
63. The method of claim 49, wherein S2 comprises GGA and F2 comprises the amino acid sequence QSGHLQR.
64. The method of claim 49, wherein S3 comprises GGA and F3 comprises the amino acid sequence QSGHLQR.
65. The method of claim 49, wherein SI comprises GGG and FI comprises the amino acid sequence RSDHLAR.
66. The method of claim 49, wherein S2 comprises GGG and F2 comprises the amino acid sequence RSDHLSR.
67. The method of claim 49, wherein S3 comprises GGG and F3 comprises the amino acid sequence RSDHLSR.
68. The method of claim 49, wherein SI comprises GGC and FI comprises the amino acid sequence DRSHLTR.
69. The method of claim 49, wherein S2 comprises GGC and F2 comprises the amino acid sequence DRSHLAR.
70. The method of claim 49, wherein SI comprises GGT and FI comprises the amino acid sequence QSSHLTR.
71. The method of claim 49, wherein S2 comprises GGT and F2 comprises the amino acid sequence TSGHLSR.
72. The method of claim 49, wherein S3 comprises GGT and F3 comprises the amino acid sequence TSGHLNR.
73. The method of claim 49, wherein SI comprises GCA and FI comprises the amino acid sequence QSGSLTR.
74. The method of claim 49, wherein S2 comprises GCA and F2 comprises the amino acid sequence QSGDLTR.
75. The method of claim 49, wherein S3 comprises GCA and F3 comprises the amino acid sequence QSGDLTR.
76. The method of claim 49, wherein SI comprises GCG and FI comprises the amino acid sequence RSDDLTR.
77. The method of claim 49, wherein S2 comprises GCG and F2 comprises the amino acid sequence RSDDLQR.
78. The method of claim 49, wherein S3 comprises GCG and F3 comprises the amino acid sequence RSDDLTR.
79. The method of claim 49, wherein SI comprises GCC and FI comprises the amino acid sequence ERGTLAR.
80. The method of claim 49, wherein S2 comprises GCC and F2 comprises the amino acid sequence DRSDLTR.
81. The method of claim 49, wherein S3 comprises GCC and F3 comprises the amino acid sequence DRSDLTR.
82. The method of claim 49, wherein SI comprises GCT and FI comprises the amino acid sequence QSSDLTR.
83. The method of claim 49, wherein S2 comprises GCT and F2 comprises the amino acid sequence QSSDLTR.
84. The method of claim 49, wherein S3 comprises GCT and F3 comprises the amino acid sequence QSSDLQR.
85. The method of claim 49, wherein SI comprises GTA and FI comprises the amino acid sequence QSGALTR.
86. The method of claim 49, wherein S2 comprises GTA and F2 comprises the amino acid sequence QSGALAR.
87. The method of claim 49, wherein SI comprises GTG and FI comprises the amino acid sequence RSDALTR.
88. The method of claim 49, wherein S2 comprises GTG and F2 comprises the amino acid sequence RSDALSR.
89. The method of claim 49, wherein S3 comprises GTG and F3 comprises the amino acid sequence RSDALTR.
90. The method of claim 49, wherein SI comprises GTC and FI comprises the amino acid sequence DRSALAR.
91. The method of claim 49, wherein S2 comprises GTC and F2 comprises the amino acid sequence DRSALAR.
92. The method of claim 49, wherein S3 comprises GTC and F3 comprises the amino acid sequence DRSALAR.
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Families Citing this family (306)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030104526A1 (en) 1999-03-24 2003-06-05 Qiang Liu Position dependent recognition of GNN nucleotide triplets by zinc fingers
US7947469B2 (en) 2001-01-22 2011-05-24 Gendaq, Ltd. Modulation of HIV infection
US20070178454A1 (en) * 2002-10-21 2007-08-02 Joung J K Context sensitive paralell optimization of zinc finger dna binding domains
EP1651660B1 (en) 2003-08-08 2018-01-24 Sangamo Therapeutics, Inc. Methods and compositions for targeted cleavage and recombination
US20120196370A1 (en) 2010-12-03 2012-08-02 Fyodor Urnov Methods and compositions for targeted genomic deletion
US7888121B2 (en) 2003-08-08 2011-02-15 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
ATE518960T1 (en) 2003-09-19 2011-08-15 Sangamo Biosciences Inc GENETICALLY PRODUCED ZINC FINGER PROTEINS TO REGULATE GENE EXPRESSION
US7534775B2 (en) * 2004-04-08 2009-05-19 Sangamo Biosciences, Inc. Methods and compositions for modulating cardiac contractility
KR20070060115A (en) 2004-09-16 2007-06-12 상가모 바이오사이언스 인코포레이티드 Compositions and methods for protein production
ATE553122T1 (en) * 2005-02-28 2012-04-15 Sangamo Biosciences Inc ANTIANGIOGENIC METHODS AND COMPOSITIONS
KR20080033455A (en) 2005-07-26 2008-04-16 상가모 바이오사이언스 인코포레이티드 Targeted integration and expression of exogenous nucleic acid sequences
US7919583B2 (en) * 2005-08-08 2011-04-05 Discovery Genomics, Inc. Integration-site directed vector systems
EP2341135A3 (en) 2005-10-18 2011-10-12 Precision Biosciences Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity
WO2009134714A2 (en) * 2008-04-28 2009-11-05 Precision Biosciences, Inc. Fusion molecules of rationally-designed dna-binding proteins and effector domains
WO2007106603A2 (en) * 2006-01-06 2007-09-20 The Scripps Research Institute Specific labeling of proteins with zinc finger tags and use of zinc-finger-tagged proteins for analysis
JP2009537140A (en) * 2006-05-19 2009-10-29 サンガモ バイオサイエンシーズ， インコーポレイテッド Methods and compositions for inactivation of dihydrofolate reductase
EP2019839B1 (en) 2006-05-25 2011-12-07 Sangamo BioSciences, Inc. Methods and compositions for gene inactivation
US9428756B2 (en) 2006-08-11 2016-08-30 Dow Agrosciences Llc Zinc finger nuclease-mediated homologous recombination
EP2089427B1 (en) 2006-11-13 2014-07-30 Sangamo BioSciences, Inc. Zinc finger nuclease for targeting the human glucocorticoid receptor locus
AU2007334468B2 (en) * 2006-12-14 2013-02-07 Corteva Agriscience Llc Optimized non-canonical zinc finger proteins
CA2684378C (en) 2007-04-26 2016-11-29 Sangamo Biosciences, Inc. Targeted integration into the ppp1r12c locus
EP2167666A2 (en) 2007-06-29 2010-03-31 Pioneer Hi-Bred International Inc. Methods for altering the genome of a monocot plant cell
CA2692453C (en) 2007-07-12 2018-01-09 Trevor Collingwood Methods and compositions for inactivating alpha 1,6 fucosyltransferase (fut8) gene expression
US11235026B2 (en) 2007-09-27 2022-02-01 Sangamo Therapeutics, Inc. Methods and compositions for modulating PD1
US8563314B2 (en) 2007-09-27 2013-10-22 Sangamo Biosciences, Inc. Methods and compositions for modulating PD1
WO2009042163A2 (en) 2007-09-27 2009-04-02 Sangamo Biosciences, Inc. Rapid in vivo identification of biologically active nucleases
US8399218B2 (en) 2007-09-27 2013-03-19 Dow Agrosciences, Llc Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes
US8936936B2 (en) 2007-10-25 2015-01-20 Sangamo Biosciences, Inc. Methods and compositions for targeted integration
WO2009076292A2 (en) * 2007-12-07 2009-06-18 Precision Biosciences, Inc. Rationally-designed meganucleases with recognition sequences found in dnase hypersensitive regions of the human genome
CA2714378A1 (en) * 2008-02-08 2009-08-13 Sangamo Biosciences, Inc. Treatment of chronic pain with zinc finger proteins
WO2009114321A2 (en) * 2008-03-11 2009-09-17 Precision Biosciencs, Inc. Rationally-designed meganucleases for maize genome engineering
US20100071083A1 (en) * 2008-03-12 2010-03-18 Smith James J Temperature-dependent meganuclease activity
EP2281050B1 (en) 2008-04-14 2014-04-02 Sangamo BioSciences, Inc. Linear donor constructs for targeted integration
PL2274430T3 (en) * 2008-04-30 2017-09-29 Sanbio Inc Neural regenerating cells with alterations in dna methylation
JP2011521643A (en) 2008-05-28 2011-07-28 サンガモ バイオサイエンシーズ， インコーポレイテッド Composition for linking a DNA binding domain and a cleavage domain
EP2130836B1 (en) * 2008-06-03 2013-02-13 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Means and methods for producing zinc fingers and concatemers thereof
JP5763530B2 (en) 2008-06-10 2015-08-12 サンガモ バイオサイエンシーズ， インコーポレイテッド Methods and compositions for the generation of Bax- and Bak-deficient cell lines
CA2730921A1 (en) * 2008-07-14 2010-01-21 Precision Biosciences, Inc. Recognition sequences for i-crei-derived meganucleases and uses thereof
KR20160015400A (en) 2008-08-22 2016-02-12 상가모 바이오사이언스 인코포레이티드 Methods and compositions for targeted single-stranded cleavage and targeted integration
US8153399B2 (en) 2008-10-29 2012-04-10 Sangamo Biosciences, Inc. Methods and compositions for inactivating glutamine synthetase gene expression
CA2745031C (en) 2008-12-04 2018-08-14 Sangamo Biosciences, Inc. Genome editing in rats using zinc-finger nucleases
PT2370575T (en) 2008-12-17 2017-12-11 Dow Agrosciences Llc Targeted integration into the zp15 locus
EP3354275B1 (en) 2009-02-04 2019-10-30 Sangamo Therapeutics, Inc. Methods and compositions for treating neuropathies
US8871905B2 (en) 2009-03-20 2014-10-28 Sangamo Biosciences, Inc. Modification of CXCR4 using engineered zinc finger proteins
AU2010235161B2 (en) 2009-04-09 2015-01-22 Sangamo Therapeutics, Inc. Targeted integration into stem cells
US8772008B2 (en) 2009-05-18 2014-07-08 Sangamo Biosciences, Inc. Methods and compositions for increasing nuclease activity
JP5798116B2 (en) 2009-06-30 2015-10-21 サンガモ バイオサイエンシーズ， インコーポレイテッド Rapid screening of biologically active nucleases and isolation of nuclease modified cells
US20120171771A1 (en) 2009-07-08 2012-07-05 Cellular Dynamics International, Inc. Modified ips cells having a mutant form of a human immunodeficiency virus (hiv) cellular entry gene
EP2461819A4 (en) * 2009-07-28 2013-07-31 Sangamo Biosciences Inc Methods and compositions for treating trinucleotide repeat disorders
EP2462230B1 (en) * 2009-08-03 2015-07-15 Recombinetics, Inc. Methods and compositions for targeted gene modification
US20110041195A1 (en) 2009-08-11 2011-02-17 Sangamo Biosciences, Inc. Organisms homozygous for targeted modification
NZ619018A (en) * 2009-10-22 2014-06-27 Dow Agrosciences Llc Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis
US8956828B2 (en) 2009-11-10 2015-02-17 Sangamo Biosciences, Inc. Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases
US9420770B2 (en) 2009-12-01 2016-08-23 Indiana University Research & Technology Corporation Methods of modulating thrombocytopenia and modified transgenic pigs
AU2010339481B2 (en) 2009-12-30 2016-02-04 Pioneer Hi-Bred International, Inc. Methods and compositions for targeted polynucleotide modification
AU2011207769B2 (en) 2010-01-22 2015-05-28 Corteva Agriscience Llc Targeted genomic alteration
US9255259B2 (en) 2010-02-09 2016-02-09 Sangamo Biosciences, Inc. Targeted genomic modification with partially single-stranded donor molecules
WO2011139335A1 (en) 2010-04-26 2011-11-10 Sangamo Biosciences, Inc. Genome editing of a rosa locus using zinc-finger nucleases
PL2566972T3 (en) 2010-05-03 2020-06-29 Sangamo Therapeutics, Inc. Compositions for linking zinc finger modules
AU2011281062B2 (en) 2010-07-21 2015-01-22 Board Of Regents, The University Of Texas System Methods and compositions for modification of a HLA locus
JP2013537410A (en) 2010-07-23 2013-10-03 シグマ−アルドリッチ・カンパニー・リミテッド・ライアビリティ・カンパニー Genome editing using targeted endonucleases and single-stranded nucleic acids
WO2012047598A1 (en) 2010-09-27 2012-04-12 Sangamo Biosciences, Inc. Methods and compositions for inhibiting viral entry into cells
KR101930711B1 (en) 2010-10-12 2018-12-19 더 칠드런스 호스피탈 오브 필라델피아 Methods and compositions for treating hemophilia b
CA2821547A1 (en) 2010-12-29 2012-07-05 Sigma-Aldrich Co. Llc Cells having disrupted expression of proteins involved in adme and toxicology processes
WO2012094132A1 (en) 2011-01-05 2012-07-12 Sangamo Biosciences, Inc. Methods and compositions for gene correction
WO2012129373A2 (en) 2011-03-23 2012-09-27 Pioneer Hi-Bred International, Inc. Methods for producing a complex transgenic trait locus
EP2694554A1 (en) 2011-04-08 2014-02-12 Gilead Biologics, Inc. Methods and compositions for normalization of tumor vasculature by inhibition of loxl2
JP6158170B2 (en) 2011-04-27 2017-07-12 アミリス， インコーポレイテッド Methods for genome modification
WO2013066423A2 (en) 2011-06-21 2013-05-10 Pioneer Hi-Bred International, Inc. Methods and compositions for producing male sterile plants
BR112013033448B1 (en) 2011-06-30 2022-01-25 Sigma-Aldrich Co. Llc Method for producing a recombinant protein with a human-like glycosylation pattern
EP2737063B1 (en) 2011-07-25 2016-06-01 Sangamo BioSciences, Inc. Methods and compositions for alteration of a cystic fibrosis transmembrane conductance regulator (cftr) gene
US9150847B2 (en) 2011-09-21 2015-10-06 Sangamo Biosciences, Inc. Methods and compositions for regulation of transgene expression
JP6188703B2 (en) 2011-10-27 2017-08-30 サンガモ セラピューティクス， インコーポレイテッド Methods and compositions for modifying the HPRT locus
CA2854819C (en) 2011-11-16 2022-07-19 Sangamo Biosciences, Inc. Modified dna-binding proteins and uses thereof
JP6262665B2 (en) 2012-01-11 2018-01-17 シグマ−アルドリッチ・カンパニー、エルエルシー Production of recombinant proteins with simple glycoforms
EP4218727A3 (en) 2012-01-27 2023-08-30 SanBio, Inc. Methods and compositions for modulating angiogenesis and vasculogenesis
EP2820127B1 (en) 2012-02-28 2018-09-26 Sigma Aldrich Co. LLC Targeted histone acetylation
MX359327B (en) 2012-02-29 2018-09-25 Sangamo Biosciences Inc Methods and compositions for treating huntington's disease.
KR102091298B1 (en) 2012-05-02 2020-03-19 다우 아그로사이언시즈 엘엘씨 Targeted modification of malate dehydrogenase
EP2847338B1 (en) 2012-05-07 2018-09-19 Sangamo Therapeutics, Inc. Methods and compositions for nuclease-mediated targeted integration of transgenes
US10648001B2 (en) 2012-07-11 2020-05-12 Sangamo Therapeutics, Inc. Method of treating mucopolysaccharidosis type I or II
HUE051612T2 (en) 2012-07-11 2021-03-01 Sangamo Therapeutics Inc Methods and compositions for the treatment of lysosomal storage diseases
EP2872154B1 (en) 2012-07-11 2017-05-31 Sangamo BioSciences, Inc. Methods and compositions for delivery of biologics
IN2015DN01480A (en) 2012-08-29 2015-07-03 Sangamo Biosciences Inc
DK2893022T3 (en) 2012-09-04 2020-07-27 Scripps Research Inst CHIMERIC POLYPEPTIDES WITH TARGETED BINDING SPECIFICITY
US9937205B2 (en) 2012-09-04 2018-04-10 The Trustees Of The University Of Pennsylvania Inhibition of diacylglycerol kinase to augment adoptive T cell transfer
UA118090C2 (en) 2012-09-07 2018-11-26 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
CN105264067B (en) 2012-09-07 2020-11-10 美国陶氏益农公司 FAD3 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
UA119135C2 (en) 2012-09-07 2019-05-10 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Engineered transgene integration platform (etip) for gene targeting and trait stacking
AU2013329186B2 (en) 2012-10-10 2019-02-14 Sangamo Therapeutics, Inc. T cell modifying compounds and uses thereof
JP2016500254A (en) 2012-12-05 2016-01-12 サンガモ バイオサイエンシーズ， インコーポレイテッド Methods and compositions for the regulation of metabolic diseases
JP2016500268A (en) 2012-12-13 2016-01-12 ダウ アグロサイエンシィズ エルエルシー Precise gene targeting for specific loci in maize
JP6491113B2 (en) 2013-02-25 2019-03-27 サンガモ セラピューティクス， インコーポレイテッド Methods and compositions for enhancing nuclease-mediated gene disruption
AU2014235968B2 (en) 2013-03-21 2018-05-24 Ospedale San Raffaele Srl Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases
RU2723130C2 (en) 2013-04-05 2020-06-08 ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи Methods and compositions for embedding an exogenous sequence into a plant genome
US10604771B2 (en) 2013-05-10 2020-03-31 Sangamo Therapeutics, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
JP2016521975A (en) 2013-05-15 2016-07-28 サンガモ バイオサイエンシーズ， インコーポレイテッド Methods and compositions for the treatment of genetic conditions
US9567609B2 (en) 2013-08-28 2017-02-14 Sangamo Biosciences, Inc. Compositions for linking DNA-binding domains and cleavage domains
US10117899B2 (en) 2013-10-17 2018-11-06 Sangamo Therapeutics, Inc. Delivery methods and compositions for nuclease-mediated genome engineering in hematopoietic stem cells
EP3058072B1 (en) 2013-10-17 2021-05-19 Sangamo Therapeutics, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
NZ719494A (en) 2013-11-04 2017-09-29 Dow Agrosciences Llc Optimal maize loci
UY35814A (en) 2013-11-04 2015-05-29 Dow Agrosciences Llc ? OPTIMAL PLACES FOR SOYBEAN ?.
RU2709734C2 (en) 2013-11-04 2019-12-19 ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи Optimum maize loci
AU2014341934B2 (en) 2013-11-04 2017-12-07 Corteva Agriscience Llc Optimal soybean loci
MX2016006106A (en) 2013-11-11 2016-12-09 Sangamo Biosciences Inc Methods and compositions for treating huntington's disease.
EP3068881B1 (en) 2013-11-13 2019-02-27 Children's Medical Center Corporation Nuclease-mediated regulation of gene expression
AU2014364051B2 (en) * 2013-12-09 2018-12-20 Sangamo Therapeutics, Inc. Methods and compositions for treating hemophilia
KR102274445B1 (en) 2013-12-19 2021-07-08 아미리스 인코퍼레이티드 Methods for genomic integration
US10072066B2 (en) 2014-02-03 2018-09-11 Sangamo Therapeutics, Inc. Methods and compositions for treatment of a beta thalessemia
WO2015127439A1 (en) 2014-02-24 2015-08-27 Sangamo Biosciences, Inc. Methods and compositions for nuclease-mediated targeted integration
TW201538518A (en) 2014-02-28 2015-10-16 Dow Agrosciences Llc Root specific expression conferred by chimeric gene regulatory elements
CN111849921A (en) 2014-03-04 2020-10-30 西格马-奥尔德里奇有限责任公司 Antiviral cells and uses thereof
JP6594891B2 (en) 2014-03-18 2019-10-23 サンガモ セラピューティクス， インコーポレイテッド Methods and compositions for modulating zinc finger protein expression
AU2015259191B2 (en) 2014-05-13 2019-03-21 Sangamo Therapeutics, Inc. Methods and compositions for prevention or treatment of a disease
WO2015188065A1 (en) 2014-06-05 2015-12-10 Sangamo Biosciences, Inc. Methods and compositions for nuclease design
WO2016005449A1 (en) 2014-07-08 2016-01-14 Vib Vzw Means and methods to increase plant yield
FI3169778T3 (en) 2014-07-14 2023-12-13 Univ Washington State Nanos knock-out that ablates germline cells
MA40253A (en) 2014-07-15 2017-05-24 Juno Therapeutics Inc Engineered cells for adoptive cell therapy
WO2016014794A1 (en) 2014-07-25 2016-01-28 Sangamo Biosciences, Inc. Methods and compositions for modulating nuclease-mediated genome engineering in hematopoietic stem cells
US9757420B2 (en) 2014-07-25 2017-09-12 Sangamo Therapeutics, Inc. Gene editing for HIV gene therapy
US9616090B2 (en) 2014-07-30 2017-04-11 Sangamo Biosciences, Inc. Gene correction of SCID-related genes in hematopoietic stem and progenitor cells
CN106795488B (en) 2014-09-16 2021-03-30 桑格摩治疗股份有限公司 Methods and compositions for nuclease-mediated genome engineering and correction in hematopoietic stem cells
US10889834B2 (en) 2014-12-15 2021-01-12 Sangamo Therapeutics, Inc. Methods and compositions for enhancing targeted transgene integration
US20180002379A1 (en) 2015-01-21 2018-01-04 Sangamo Therapeutics, Inc. Methods and compositions for identification of highly specific nucleases
AU2016243052C1 (en) 2015-04-03 2022-11-24 Dana-Farber Cancer Institute, Inc. Composition and methods of genome editing of B-cells
MX2017012936A (en) 2015-04-15 2018-02-01 Dow Agrosciences Llc Plant promoter for transgene expression.
AU2016250064A1 (en) 2015-04-15 2017-10-19 Dow Agrosciences Llc Plant promoter for transgene expression
US10179918B2 (en) 2015-05-07 2019-01-15 Sangamo Therapeutics, Inc. Methods and compositions for increasing transgene activity
IL284707B2 (en) 2015-05-12 2024-01-01 Sangamo Therapeutics Inc Nuclease-mediated regulation of gene expression
US9957501B2 (en) 2015-06-18 2018-05-01 Sangamo Therapeutics, Inc. Nuclease-mediated regulation of gene expression
US10450585B2 (en) 2015-07-13 2019-10-22 Sangamo Therapeutics, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
MA42895A (en) 2015-07-15 2018-05-23 Juno Therapeutics Inc MODIFIED CELLS FOR ADOPTIVE CELL THERAPY
BR112018002417A2 (en) 2015-08-06 2018-09-18 Univ Missouri non-human animal or descendant thereof or an animal cell, swine animal, descendant or cell, cell, descendant, reproduction method, animal population, method for increasing an animal resistance, nucleic acid molecule, nucleic acid and nucleic acid isolated
AU2016308339A1 (en) 2015-08-18 2018-04-12 Baylor College Of Medicine Methods and compositions for altering function and structure of chromatin loops and/or domains
TW201718861A (en) 2015-09-22 2017-06-01 道禮責任有限公司 Plant promoter and 3'UTR for transgene expression
TW201718862A (en) 2015-09-22 2017-06-01 Dow Agrosciences Llc Plant promoter and 3' UTR for transgene expression
CA3002151A1 (en) 2015-10-22 2017-04-27 Dow Agrosciences Llc Plant promoter for transgene expression
WO2017078935A1 (en) 2015-11-04 2017-05-11 Dow Agrosciences Llc Plant promoter for transgene expression
AU2016355178B9 (en) 2015-11-19 2019-05-30 Massachusetts Institute Of Technology Lymphocyte antigen CD5-like (CD5L)-interleukin 12B (p40) heterodimers in immunity
EP3380622A4 (en) 2015-11-23 2019-08-07 Sangamo Therapeutics, Inc. Methods and compositions for engineering immunity
CA3008382A1 (en) 2015-12-18 2017-06-22 Sangamo Therapeutics, Inc. Targeted disruption of the mhc cell receptor
CN108778297B (en) 2015-12-18 2024-02-09 桑格摩生物治疗股份有限公司 Targeted disruption of T cell receptors
EP3901258A1 (en) 2016-01-11 2021-10-27 The Board of Trustees of the Leland Stanford Junior University Chimeric proteins and methods of immunotherapy
JP7015239B2 (en) 2016-01-11 2022-03-04 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー How to regulate chimeric protein and gene expression
EP3402533B1 (en) 2016-01-15 2021-08-04 Sangamo Therapeutics, Inc. Methods and compositions for the treatment of neurologic disease
EP3769775A3 (en) 2016-02-02 2021-03-17 Sangamo Therapeutics, Inc. Compositions for linking dna-binding domains and cleavage domains
EP3417061B1 (en) 2016-02-18 2022-10-26 The Regents of the University of California Methods and compositions for gene editing in stem cells
EP3429567B1 (en) 2016-03-16 2024-01-10 The J. David Gladstone Institutes Methods and compositions for treating obesity and/or diabetes and for identifying candidate treatment agents
US11293033B2 (en) 2016-05-18 2022-04-05 Amyris, Inc. Compositions and methods for genomic integration of nucleic acids into exogenous landing pads
EP3464323B1 (en) 2016-05-27 2021-09-22 Aadigen, LLC Peptides and nanoparticles for intracellular delivery of genome-editing molecules
EP3474849A4 (en) 2016-06-27 2020-07-29 The Broad Institute, Inc. Compositions and methods for detecting and treating diabetes
ES2938210T3 (en) 2016-07-13 2023-04-05 Vertex Pharma Methods, compositions and kits to increase the efficiency of genome editing
EP3485023B1 (en) 2016-07-15 2023-11-15 Salk Institute for Biological Studies Methods and compositions for genome editing in non-dividing cells
JP2019520844A (en) 2016-07-21 2019-07-25 マックスサイト インコーポレーティッド Methods and compositions for modifying genomic DNA
US11193136B2 (en) 2016-08-09 2021-12-07 Vib Vzw Cellulose synthase inhibitors and mutant plants
PL3504229T3 (en) 2016-08-24 2022-04-19 Sangamo Therapeutics, Inc. Regulation of gene expression using engineered nucleases
SG11201901364VA (en) 2016-08-24 2019-03-28 Sangamo Therapeutics Inc Engineered target specific nucleases
WO2018049009A2 (en) 2016-09-07 2018-03-15 Sangamo Therapeutics, Inc. Modulation of liver genes
US10400246B2 (en) 2016-10-03 2019-09-03 Dow Agrosciences Llc Plant promoter for transgene expression
BR112019005687A2 (en) 2016-10-03 2019-07-02 Dow Agrosciences Llc plant promoter for transgenic expression
WO2018067826A1 (en) 2016-10-05 2018-04-12 Cellular Dynamics International, Inc. Generating mature lineages from induced pluripotent stem cells with mecp2 disruption
AU2017343780B2 (en) 2016-10-13 2023-08-31 Juno Therapeutics, Inc. Immunotherapy methods and compositions involving tryptophan metabolic pathway modulators
GB201617559D0 (en) 2016-10-17 2016-11-30 University Court Of The University Of Edinburgh The Swine comprising modified cd163 and associated methods
JP7042263B2 (en) 2016-10-20 2022-03-25 サンガモ セラピューティクス， インコーポレイテッド Methods and compositions for the treatment of Fabry disease
CA3041668A1 (en) 2016-10-31 2018-05-03 Sangamo Therapeutics, Inc. Gene correction of scid-related genes in hematopoietic stem and progenitor cells
US20190264218A1 (en) 2016-11-04 2019-08-29 Flagship Pioneering Innovations V, Inc. Novel Plant Cells, Plants, and Seeds
US11793833B2 (en) 2016-12-02 2023-10-24 Juno Therapeutics, Inc. Engineered B cells and related compositions and methods
CN110249051A (en) 2016-12-08 2019-09-17 凯斯西储大学 Enhance the method and composition that functional myelin generates
KR20240027888A (en) 2016-12-14 2024-03-04 리간달 인코포레이티드 Methods and compositions for nucleic acid and protein payload delivery
WO2018140478A1 (en) 2017-01-24 2018-08-02 Sigma-Aldrich Co. Llc Viral resistant cells and culture systems
US20210115407A1 (en) 2017-04-12 2021-04-22 The Broad Institute, Inc. Respiratory and sweat gland ionocytes
US20200071773A1 (en) 2017-04-12 2020-03-05 Massachusetts Eye And Ear Infirmary Tumor signature for metastasis, compositions of matter methods of use thereof
WO2018191657A1 (en) 2017-04-13 2018-10-18 Acuitas Therapeutics, Inc. Lipids for delivery of active agents
US20200384115A1 (en) 2017-04-21 2020-12-10 The Broad Institute , Inc. Targeted delivery to beta cells
AU2018256877B2 (en) 2017-04-28 2022-06-02 Acuitas Therapeutics, Inc. Novel carbonyl lipids and lipid nanoparticle formulations for delivery of nucleic acids
CA3059793A1 (en) 2017-05-03 2018-11-08 Sangamo Therapeutics, Inc. Methods and compositions for modification of a cystic fibrosis transmembrane conductance regulator (cftr) gene
WO2018204777A2 (en) 2017-05-05 2018-11-08 The Broad Institute, Inc. Methods for identification and modification of lncrna associated with target genotypes and phenotypes
US11897953B2 (en) 2017-06-14 2024-02-13 The Broad Institute, Inc. Compositions and methods targeting complement component 3 for inhibiting tumor growth
US11512287B2 (en) 2017-06-16 2022-11-29 Sangamo Therapeutics, Inc. Targeted disruption of T cell and/or HLA receptors
WO2019005884A1 (en) 2017-06-26 2019-01-03 The Broad Institute, Inc. Crispr/cas-adenine deaminase based compositions, systems, and methods for targeted nucleic acid editing
US20200255828A1 (en) 2017-10-04 2020-08-13 The Broad Institute, Inc. Methods and compositions for altering function and structure of chromatin loops and/or domains
KR20200079539A (en) 2017-11-09 2020-07-03 상가모 테라퓨틱스, 인코포레이티드 Genetic modification of cytokine-inducible SH2-containing protein (CISH) gene
US20210363260A1 (en) 2017-11-13 2021-11-25 The Broad Institute, Inc. Methods and compositions for treating cancer by targeting the clec2d-klrb1 pathway
SG11202004457XA (en) 2017-11-17 2020-06-29 Iovance Biotherapeutics Inc Til expansion from fine needle aspirates and small biopsies
EP3714041A1 (en) 2017-11-22 2020-09-30 Iovance Biotherapeutics, Inc. Expansion of peripheral blood lymphocytes (pbls) from peripheral blood
EA202091709A1 (en) 2018-01-17 2020-11-10 Вертекс Фармасьютикалз Инкорпорейтед DNA PC INHIBITORS
JP7391854B2 (en) 2018-01-17 2023-12-05 バーテックス ファーマシューティカルズ インコーポレイテッド DNA-PK inhibitor
JP2021511312A (en) 2018-01-17 2021-05-06 バーテックス ファーマシューティカルズ インコーポレイテッドＶｅｒｔｅｘ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ Ｉｎｃｏｒｐｏｒａｔｅｄ Kinoxalinone compounds, compositions, methods, and kits for increasing genome editing efficiency
WO2019157324A1 (en) 2018-02-08 2019-08-15 Sangamo Therapeutics, Inc. Engineered target specific nucleases
WO2019195738A1 (en) 2018-04-06 2019-10-10 Children's Medical Center Corporation Compositions and methods for somatic cell reprogramming and modulating imprinting
US11421007B2 (en) 2018-04-18 2022-08-23 Sangamo Therapeutics, Inc. Zinc finger protein compositions for modulation of huntingtin (Htt)
ES2922902T3 (en) 2018-04-24 2022-09-21 Kws Saat Se & Co Kgaa Plants with improved digestibility and marker haplotypes
WO2019210268A2 (en) 2018-04-27 2019-10-31 The Broad Institute, Inc. Sequencing-based proteomics
MA52533A (en) 2018-04-27 2021-03-03 Iovance Biotherapeutics Inc CLOSED CIRCUIT PROCESS FOR THE AMPLIFICATION AND EDITING OF TUMOR INFILTRATION LYMPHOCYTE GENES AND THEIR USES IN IMMUNOTHERAPY
US20210386829A1 (en) 2018-05-04 2021-12-16 The Broad Institute, Inc. Compositions and methods for modulating cgrp signaling to regulate innate lymphoid cell inflammatory responses
US11690921B2 (en) 2018-05-18 2023-07-04 Sangamo Therapeutics, Inc. Delivery of target specific nucleases
WO2019232542A2 (en) 2018-06-01 2019-12-05 Massachusetts Institute Of Technology Methods and compositions for detecting and modulating microenvironment gene signatures from the csf of metastasis patients
US20210317429A1 (en) 2018-08-20 2021-10-14 The Broad Institute, Inc. Methods and compositions for optochemical control of crispr-cas9
KR20210049124A (en) 2018-08-23 2021-05-04 상가모 테라퓨틱스, 인코포레이티드 Engineered target specific base editor
WO2020051283A1 (en) 2018-09-05 2020-03-12 The Regents Of The University Of California Generation of heritably gene-edited plants without tissue culture
EP3849565A4 (en) 2018-09-12 2022-12-28 Fred Hutchinson Cancer Research Center Reducing cd33 expression to selectively protect therapeutic cells
CN113015741A (en) 2018-09-18 2021-06-22 桑格摩生物治疗股份有限公司 Programmed cell death 1(PD1) specific nucleases
JP7344300B2 (en) 2018-09-18 2023-09-13 ブイエヌブイ ニューコ インク． ARC-based capsids and their uses
WO2020077236A1 (en) 2018-10-12 2020-04-16 The Broad Institute, Inc. Method for extracting nuclei or whole cells from formalin-fixed paraffin-embedded tissues
WO2020079033A1 (en) 2018-10-15 2020-04-23 Fondazione Telethon Genome editing methods and constructs
WO2020081730A2 (en) 2018-10-16 2020-04-23 Massachusetts Institute Of Technology Methods and compositions for modulating microenvironment
JOP20210094A1 (en) 2018-11-05 2023-01-30 Iovance Biotherapeutics Inc Processes for production of tumor infiltrating lymphocytes and uses of the same in immunotherapy
KR20210091212A (en) 2018-11-05 2021-07-21 이오반스 바이오테라퓨틱스, 인크. Treatment of NSCLC Patients Refractory to Anti-PD-1 Antibodies
CN113272420A (en) 2018-11-05 2021-08-17 艾欧凡斯生物治疗公司 Improved selection of tumor-reactive T cells
US20220033775A1 (en) 2018-11-05 2022-02-03 Iovance Biotherapeutics, Inc. Expansion of tils utilizing akt pathways inhibitors
EP3898958A1 (en) 2018-12-17 2021-10-27 The Broad Institute, Inc. Crispr-associated transposase systems and methods of use thereof
US20220193131A1 (en) 2018-12-19 2022-06-23 Iovance Biotherapeutics, Inc. Methods of Expanding Tumor Infiltrating Lymphocytes Using Engineered Cytokine Receptor Pairs and Uses Thereof
US11739156B2 (en) 2019-01-06 2023-08-29 The Broad Institute, Inc. Massachusetts Institute of Technology Methods and compositions for overcoming immunosuppression
SG11202106987WA (en) 2019-01-11 2021-07-29 Acuitas Therapeutics Inc Lipids for lipid nanoparticle delivery of active agents
CA3106693A1 (en) 2019-02-06 2020-08-13 Sangamo Therapeutics, Inc. Method for the treatment of mucopolysaccharidosis type i
WO2020163856A1 (en) 2019-02-10 2020-08-13 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Modified mitochondrion and methods of use thereof
WO2020172343A2 (en) 2019-02-19 2020-08-27 Massachusetts Institute Of Technology Methods for treating injuries
JP2022522473A (en) 2019-03-01 2022-04-19 アイオバンス バイオセラピューティクス，インコーポレイテッド Expansion culture of tumor-infiltrating lymphocytes from humoral tumors and their therapeutic use
US20220154282A1 (en) 2019-03-12 2022-05-19 The Broad Institute, Inc. Detection means, compositions and methods for modulating synovial sarcoma cells
US20220152115A1 (en) 2019-03-13 2022-05-19 The Broad Institute, Inc. Microglial progenitors for regeneration of functional microglia in the central nervous system and therapeutics uses thereof
WO2020186235A1 (en) 2019-03-14 2020-09-17 The Broad Institute, Inc. Compositions and methods for modulating cgrp signaling to regulate intestinal innate lymphoid cells
US20220142948A1 (en) 2019-03-18 2022-05-12 The Broad Institute, Inc. Compositions and methods for modulating metabolic regulators of t cell pathogenicity
WO2020191069A1 (en) 2019-03-18 2020-09-24 The Broad Institute, Inc. Modulation of type 2 immunity by targeting clec-2 signaling
MA55531A (en) 2019-04-02 2022-02-09 Sangamo Therapeutics Inc METHODS FOR THE TREATMENT OF BETA-THALASSEMIA
CN113966401A (en) 2019-04-10 2022-01-21 犹他大学研究基金会 HTRA1 modulation for the treatment of AMD
WO2020232029A1 (en) 2019-05-13 2020-11-19 Iovance Biotherapeutics, Inc. Methods and compositions for selecting tumor infiltrating lymphocytes and uses of the same in immunotherapy
EP3969607A1 (en) 2019-05-13 2022-03-23 KWS SAAT SE & Co. KGaA Drought tolerance in corn
WO2020232271A1 (en) 2019-05-14 2020-11-19 The Broad Institute, Inc. Compositions and methods for targeting multinucleated cells
AR118995A1 (en) 2019-05-25 2021-11-17 Kws Saat Se & Co Kgaa HAPLOID INDUCTION ENHANCER
US20220226464A1 (en) 2019-05-28 2022-07-21 Massachusetts Institute Of Technology Methods and compositions for modulating immune responses
US20220243178A1 (en) 2019-05-31 2022-08-04 The Broad Institute, Inc. Methods for treating metabolic disorders by targeting adcy5
SG11202112092TA (en) 2019-06-25 2021-11-29 Inari Agriculture Technology Inc Improved homology dependent repair genome editing
WO2021022223A1 (en) 2019-08-01 2021-02-04 Sana Biotechnology, Inc. Dux4 expressing cells and uses thereof
EP3772542A1 (en) 2019-08-07 2021-02-10 KWS SAAT SE & Co. KGaA Modifying genetic variation in crops by modulating the pachytene checkpoint protein 2
WO2021030627A1 (en) 2019-08-13 2021-02-18 The General Hospital Corporation Methods for predicting outcomes of checkpoint inhibition and treatment thereof
KR20220068222A (en) 2019-08-23 2022-05-25 사나 바이오테크놀로지, 인크. CD24 expressing cells and uses thereof
US20220298501A1 (en) 2019-08-30 2022-09-22 The Broad Institute, Inc. Crispr-associated mu transposase systems
US11793787B2 (en) 2019-10-07 2023-10-24 The Broad Institute, Inc. Methods and compositions for enhancing anti-tumor immunity by targeting steroidogenesis
AU2020366566A1 (en) 2019-10-17 2022-04-21 KWS SAAT SE & Co. KGaA Enhanced disease resistance of crops by downregulation of repressor genes
US20220389381A1 (en) 2019-10-25 2022-12-08 Iovance Biotherapeutics, Inc. Gene editing of tumor infiltrating lymphocytes and uses of same in immunotherapy
US11844800B2 (en) 2019-10-30 2023-12-19 Massachusetts Institute Of Technology Methods and compositions for predicting and preventing relapse of acute lymphoblastic leukemia
WO2021087366A1 (en) 2019-11-01 2021-05-06 Sangamo Therapeutics, Inc. Compositions and methods for genome engineering
CA3161104A1 (en) 2019-12-11 2021-06-17 Cecile Chartier-Courtaud Processes for the production of tumor infiltrating lymphocytes (tils) and methods of using the same
JPWO2021117874A1 (en) 2019-12-13 2021-06-17
US20230293593A1 (en) 2020-03-25 2023-09-21 Sana Biotechnology, Inc. Hypoimmunogenic neural cells for the treatment of neurological disorders and conditions
US20230203469A1 (en) 2020-04-02 2023-06-29 Takeda Pharmaceutical Company Limited Adamts13 variant, compositions, and uses thereof
US20230172987A1 (en) 2020-05-04 2023-06-08 Iovance Biotherapeutics, Inc. Processes for production of tumor infiltrating lymphocytes and uses of the same in immunotherapy
CN115715203A (en) 2020-05-06 2023-02-24 塞勒克提斯公司 Methods of genetically modifying cells to deliver therapeutic proteins
JP2023525513A (en) 2020-05-06 2023-06-16 セレクティス ソシエテ アノニム Methods for targeted insertion of exogenous sequences in the cellular genome
WO2021224633A1 (en) 2020-05-06 2021-11-11 Orchard Therapeutics (Europe) Limited Treatment for neurodegenerative diseases
WO2021236852A1 (en) 2020-05-20 2021-11-25 Sana Biotechnology, Inc. Methods and compositions for treatment of viral infections
EP4156913A1 (en) 2020-05-29 2023-04-05 KWS SAAT SE & Co. KGaA Plant haploid induction
EP4161552A1 (en) 2020-06-05 2023-04-12 The Broad Institute, Inc. Compositions and methods for treating neoplasia
IL300516A (en) 2020-08-13 2023-04-01 Sana Biotechnology Inc Methods of treating sensitized patients with hypoimmunogenic cells, and associated methods and compositions
WO2022046760A2 (en) 2020-08-25 2022-03-03 Kite Pharma, Inc. T cells with improved functionality
WO2022076606A1 (en) 2020-10-06 2022-04-14 Iovance Biotherapeutics, Inc. Treatment of nsclc patients with tumor infiltrating lymphocyte therapies
CN117042600A (en) 2020-11-16 2023-11-10 猪改良英国有限公司 Anti-influenza a animals with edited ANP32 gene
JP2024500403A (en) 2020-12-17 2024-01-09 アイオバンス バイオセラピューティクス，インコーポレイテッド Treatment of cancer with tumor-infiltrating lymphocytes
CA3202483A1 (en) 2020-12-17 2022-06-23 Maria Fardis Treatment with tumor infiltrating lymphocyte therapies in combination with ctla-4 and pd-1 inhibitors
TW202302841A (en) 2020-12-31 2023-01-16 美商薩那生物科技公司 Methods and compositions for modulating car-t activity
TW202241508A (en) 2021-01-29 2022-11-01 美商艾歐凡斯生物治療公司 Cytokine associated tumor infiltrating lymphocytes compositions and methods
EP4308691A1 (en) 2021-03-19 2024-01-24 Iovance Biotherapeutics, Inc. Methods for tumor infiltrating lymphocyte (til) expansion related to cd39/cd69 selection and gene knockout in tils
WO2022225981A2 (en) 2021-04-19 2022-10-27 Iovance Biotherapeutics, Inc. Chimeric costimulatory receptors, chemokine receptors, and the use of same in cellular immunotherapies
CA3219148A1 (en) 2021-05-17 2022-11-24 Frederick G. Vogt Pd-1 gene-edited tumor infiltrating lymphocytes and uses of same in immunotherapy
AU2022283291A1 (en) 2021-05-27 2023-11-02 Sana Biotechnology, Inc. Hypoimmunogenic cells comprising engineered hla-e or hla-g
IL310089A (en) 2021-07-14 2024-03-01 Sana Biotechnology Inc Altered expression of y chromosome-linked antigens in hypoimmunogenic cells
WO2023009716A1 (en) 2021-07-28 2023-02-02 Iovance Biotherapeutics, Inc. Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with kras inhibitors
CA3221517A1 (en) 2021-07-30 2023-02-02 Monika KLOIBER-MAITZ Plants with improved digestibility and marker haplotypes
WO2023019229A1 (en) 2021-08-11 2023-02-16 Sana Biotechnology, Inc. Genetically modified primary cells for allogeneic cell therapy
AU2022325955A1 (en) 2021-08-11 2024-02-08 Sana Biotechnology, Inc. Genetically modified cells for allogeneic cell therapy to reduce instant blood mediated inflammatory reactions
WO2023019226A1 (en) 2021-08-11 2023-02-16 Sana Biotechnology, Inc. Genetically modified cells for allogeneic cell therapy
CA3227613A1 (en) 2021-08-11 2023-02-16 William Dowdle Inducible systems for altering gene expression in hypoimmunogenic cells
WO2023019227A1 (en) 2021-08-11 2023-02-16 Sana Biotechnology, Inc. Genetically modified cells for allogeneic cell therapy to reduce complement-mediated inflammatory reactions
US20230226116A1 (en) 2021-10-20 2023-07-20 University Of Rochester Method for rejuvenating glial progenitor cells and rejuvenated glial progenitor cells per se
AU2022370552A1 (en) 2021-10-20 2024-03-28 University Of Rochester Compositions and methods for treating myelin deficiency by rejuvenating glial progenitor cells
WO2023069790A1 (en) 2021-10-22 2023-04-27 Sana Biotechnology, Inc. Methods of engineering allogeneic t cells with a transgene in a tcr locus and associated compositions and methods
US20230187042A1 (en) 2021-10-27 2023-06-15 Iovance Biotherapeutics, Inc. Systems and methods for coordinating manufacturing of cells for patient-specific immunotherapy
WO2023081756A1 (en) 2021-11-03 2023-05-11 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Precise genome editing using retrons
TW202328201A (en) 2021-11-09 2023-07-16 美商安進公司 Method of producing an antibody peptide conjugate
WO2023093862A1 (en) 2021-11-26 2023-06-01 Epigenic Therapeutics Inc. Method of modulating pcsk9 and uses thereof
WO2023102550A2 (en) 2021-12-03 2023-06-08 The Broad Institute, Inc. Compositions and methods for efficient in vivo delivery
WO2023105244A1 (en) 2021-12-10 2023-06-15 Pig Improvement Company Uk Limited Editing tmprss2/4 for disease resistance in livestock
GB202118058D0 (en) 2021-12-14 2022-01-26 Univ Warwick Methods to increase yields in crops
WO2023122337A1 (en) 2021-12-23 2023-06-29 Sana Biotechnology, Inc. Chimeric antigen receptor (car) t cells for treating autoimmune disease and associated methods
WO2023131616A1 (en) 2022-01-05 2023-07-13 Vib Vzw Means and methods to increase abiotic stress tolerance in plants
WO2023131637A1 (en) 2022-01-06 2023-07-13 Vib Vzw Improved silage grasses
WO2023141602A2 (en) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2023144199A1 (en) 2022-01-26 2023-08-03 Vib Vzw Plants having reduced levels of bitter taste metabolites
WO2023150553A1 (en) 2022-02-01 2023-08-10 University Of Rochester Gpr17 promoter-based targeting and transduction of glial progenitor cells
WO2023150557A1 (en) 2022-02-01 2023-08-10 University Of Rochester Methods of generating a population of neurons from human glial progenitor cells and genetic constructs for carrying out such methods
WO2023154578A1 (en) 2022-02-14 2023-08-17 Sana Biotechnology, Inc. Methods of treating patients exhibiting a prior failed therapy with hypoimmunogenic cells
WO2023158836A1 (en) 2022-02-17 2023-08-24 Sana Biotechnology, Inc. Engineered cd47 proteins and uses thereof
WO2023168397A1 (en) 2022-03-04 2023-09-07 Sigma-Aldrich Co. Llc Metabolic selection via the asparagine biosynthesis pathway
WO2023173123A1 (en) 2022-03-11 2023-09-14 Sana Biotechnology, Inc. Genetically modified cells and compositions and uses thereof
WO2023196818A1 (en) 2022-04-04 2023-10-12 The Regents Of The University Of California Genetic complementation compositions and methods
WO2023196877A1 (en) 2022-04-06 2023-10-12 Iovance Biotherapeutics, Inc. Treatment of nsclc patients with tumor infiltrating lymphocyte therapies
WO2023201369A1 (en) 2022-04-15 2023-10-19 Iovance Biotherapeutics, Inc. Til expansion processes using specific cytokine combinations and/or akti treatment
WO2023213831A1 (en) 2022-05-02 2023-11-09 Fondazione Telethon Ets Homology independent targeted integration for gene editing
WO2023220608A1 (en) 2022-05-10 2023-11-16 Iovance Biotherapeutics, Inc. Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with an il-15r agonist
EP4278891A1 (en) 2022-05-20 2023-11-22 KWS SAAT SE & Co. KGaA Clubroot resistance and markers in brassica
GB2621813A (en) 2022-06-30 2024-02-28 Univ Newcastle Preventing disease recurrence in Mitochondrial replacement therapy
WO2024013514A2 (en) 2022-07-15 2024-01-18 Pig Improvement Company Uk Limited Gene edited livestock animals having coronavirus resistance
WO2024044723A1 (en) 2022-08-25 2024-02-29 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2024042199A1 (en) 2022-08-26 2024-02-29 KWS SAAT SE & Co. KGaA Use of paired genes in hybrid breeding
WO2024073692A1 (en) 2022-09-30 2024-04-04 Sigma-Aldrich Co. Llc Metabolic selection via the glycine-formate biosynthesis pathway
WO2024073686A1 (en) 2022-09-30 2024-04-04 Sigma-Aldrich Co. Llc Metabolic selection via the serine biosynthesis pathway

Family Cites Families (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3410650A1 (en) 1984-03-23 1985-10-03 Kernforschungsanlage Jülich GmbH, 5170 Jülich POROISE INORGANIC CARRIERS GROWN WITH MICRO-ORGANISMS, METHOD FOR IMMOBILIZING MICRO-ORGANISMS AND CARRIER BODIES SUITABLE FOR THIS
US5455170A (en) 1986-08-22 1995-10-03 Hoffmann-La Roche Inc. Mutated thermostable nucleic acid polymerase enzyme from Thermus species Z05
US5317090A (en) 1987-12-16 1994-05-31 Institut Pasteur Steroid/thyroid hormone receptor-related gene, which is inappropriately expressed in human hepatocellular carcinoma, and which is a retinoic acid receptor
US5324819A (en) 1988-04-08 1994-06-28 Stryker Corporation Osteogenic proteins
US5340739A (en) 1988-07-13 1994-08-23 Brigham & Women's Hospital Hematopoietic cell specific transcriptional regulatory elements of serglycin and uses thereof
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
JPH04504654A (en) 1988-12-23 1992-08-20 ザ　ソーク　インスティテュート　フォア　バイオロジカル　スタディーズ Composition and method for receptor transcription suppression activity
US5198346A (en) 1989-01-06 1993-03-30 Protein Engineering Corp. Generation and selection of novel DNA-binding proteins and polypeptides
US5096815A (en) 1989-01-06 1992-03-17 Protein Engineering Corporation Generation and selection of novel dna-binding proteins and polypeptides
US4990607A (en) 1989-03-14 1991-02-05 The Rockefeller University Alteration of gene expression in plants
AU655912B2 (en) 1989-03-17 1995-01-19 Salk Institute For Biological Studies, The Hormone response element compositions and assay
WO1991007509A1 (en) 1989-11-13 1991-05-30 Massachusetts Institute Of Technology Localization and characterization of the wilms's tumor gene
US5578482A (en) 1990-05-25 1996-11-26 Georgetown University Ligand growth factors that bind to the erbB-2 receptor protein and induce cellular responses
WO1991019813A1 (en) 1990-06-11 1991-12-26 The University Of Colorado Foundation, Inc. Nucleic acid ligands
US5270163A (en) 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
US5496938A (en) 1990-06-11 1996-03-05 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands to HIV-RT and HIV-1 rev
AU655943B2 (en) 1990-09-21 1995-01-19 Salk Institute For Biological Studies, The Functional antagonism between proto-oncoprotein c-jun and hormone receptors
US5348864A (en) 1991-01-25 1994-09-20 E. R. Squibb & Sons, Inc. Mouse vav proto-oncogene DNA and protein sequences
DE69230142T2 (en) 1991-05-15 2000-03-09 Cambridge Antibody Tech METHOD FOR PRODUCING SPECIFIC BINDING PAIRS
US5324818A (en) 1991-08-21 1994-06-28 The Regents Of The University Of Michigan Proteins useful in the regulation of κB-containing genes
US5243041A (en) 1991-08-22 1993-09-07 Fernandez Pol Jose A DNA vector with isolated CDNA gene encoding metallopanstimulin
US5302519A (en) 1991-09-09 1994-04-12 Fred Hutchinson Cancer Research Center Method of producing a Mad polypeptide
US5270170A (en) 1991-10-16 1993-12-14 Affymax Technologies N.V. Peptide library and screening method
US5792640A (en) 1992-04-03 1998-08-11 The Johns Hopkins University General method to clone hybrid restriction endonucleases using lig gene
US5436150A (en) 1992-04-03 1995-07-25 The Johns Hopkins University Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease
US5487994A (en) 1992-04-03 1996-01-30 The Johns Hopkins University Insertion and deletion mutants of FokI restriction endonuclease
US5916794A (en) 1992-04-03 1999-06-29 Johns Hopkins University Methods for inactivating target DNA and for detecting conformational change in a nucleic acid
US5356802A (en) 1992-04-03 1994-10-18 The Johns Hopkins University Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease
US5324638A (en) 1992-05-13 1994-06-28 Sloan-Kettering Institute For Cancer Research Brain transcription factor, nucleic acids encoding same and uses thereof
US6140466A (en) 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
DE69534629D1 (en) 1994-01-18 2005-12-29 Scripps Research Inst DERIVATIVES OF ZINC FINGER PROTEINS AND METHODS
AU3331595A (en) 1994-08-18 1996-03-14 Ariad Pharmaceuticals, Inc. Composite dna-binding proteins and materials and methods relating thereto
CA2196419C (en) 1994-08-20 2007-08-21 Yen Choo Improvements in or relating to binding proteins for recognition of dna
GB9824544D0 (en) 1998-11-09 1999-01-06 Medical Res Council Screening system
DE4435919C1 (en) 1994-10-07 1995-12-07 Deutsches Krebsforsch DNA encoding zinc finger protein
US5871902A (en) 1994-12-09 1999-02-16 The Gene Pool, Inc. Sequence-specific detection of nucleic acid hybrids using a DNA-binding molecule or assembly capable of discriminating perfect hybrids from non-perfect hybrids
US5702914A (en) 1994-12-21 1997-12-30 The Salk Institute For Biological Studies Use of reporter genes for retinoid receptor screening assays having novel retinoid-associated response elements
JP3817739B2 (en) 1994-12-29 2006-09-06 マサチューセッツ・インスティテュート・オブ・テクノロジー Chimeric DNA binding protein
US5789538A (en) 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
AU5445296A (en) 1995-04-12 1996-10-30 Cheng Cheng Methods for preparing dna-binding proteins
AU1707497A (en) 1996-01-23 1997-08-20 Rigel Pharmaceuticals, Inc. Methods for screening for transdominant intracellular effector peptides and rna molecules
FR2752734B1 (en) 1996-09-02 1998-11-06 Cird Galderma USE OF RETINOIDS FOR THE PREPARATION OF A MEDICAMENT FOR TREATING CONDITIONS RELATED TO VEGF OVEREXPRESSION
US5939538A (en) 1996-10-25 1999-08-17 Immusol Incorporated Methods and compositions for inhibiting HIV infection of cells by cleaving HIV co-receptor RNA
DE19718249A1 (en) 1997-04-30 1998-11-05 Basf Ag Myc-binding zinc finger proteins, their production and their use
GB9710809D0 (en) 1997-05-23 1997-07-23 Medical Res Council Nucleic acid binding proteins
GB9710807D0 (en) 1997-05-23 1997-07-23 Medical Res Council Nucleic acid binding proteins
AU755784B2 (en) 1998-01-15 2002-12-19 Ariad Pharmaceuticals, Inc. Regulation of biological events using multimeric chimeric proteins
US5972615A (en) 1998-01-21 1999-10-26 Urocor, Inc. Biomarkers and targets for diagnosis, prognosis and management of prostate disease
US6410248B1 (en) 1998-01-30 2002-06-25 Massachusetts Institute Of Technology General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites
AU2678999A (en) 1998-02-13 1999-08-30 Genetrace Systems, Inc. Use of ribozymes for functionating genes
US6205404B1 (en) 1998-02-20 2001-03-20 George S. Michaels DNA-binding proteins of the zinc-finger class
ES2341926T3 (en) 1998-03-02 2010-06-29 Massachusetts Institute Of Technology POLYPROTEINS WITH ZINC FINGERS THAT HAVE IMPROVED LINKERS.
WO1999047656A2 (en) 1998-03-17 1999-09-23 Gendaq Limited Nucleic acid binding proteins
US6140081A (en) 1998-10-16 2000-10-31 The Scripps Research Institute Zinc finger binding domains for GNN
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites

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