Three hybrid assay system
First Claim
Patent Images
1. A hybrid ligand represented by the general formula:
- R1—
Y—
R2, wherein;
(i) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridine or cyclosporin, or a derivative thereof with minor structural modifications;
(ii) Y represents a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25; and
, (iii) R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide.
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Abstract
The invention provides compositions and methods for isolating ligand binding polypeptides for a user-specified ligand, and for isolating small molecule ligands for a user-specified target polypeptide using an improved class of hybrid ligand compounds.
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Citations
66 Claims
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1. A hybrid ligand represented by the general formula:
- R1—
Y—
R2, wherein;
(i) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridine or cyclosporin, or a derivative thereof with minor structural modifications;
(ii) Y represents a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25; and
,(iii) R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
- R1—
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12. A hybrid ligand represented by the general formula:
- R1—
Y—
R2, wherein;
(i) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridine or cyclosporin, or a derivative thereof with minor structural modifications;
(ii) Y represents a linker; and
,(iii) R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide;
wherein R2 binds to or inhibits a kinase. - View Dependent Claims (13, 14, 15)
- R1—
-
16. A fusion polypeptide, comprising segments P1, Cub-Z, and RM, in an order wherein Cub-Z is closer to the N-terminus of the fusion polypeptide than RM, wherein
(i) P1 is a ligand binding polypeptide that binds to a non-peptide ligand of a hybrid ligand, which has the general formula R1— - Y—
R2, where R1 and R2 are ligands, and Y is a linker,(ii) Cub is a carboxy-terminal subdomain of ubiquitin, (iii) Z is an amino acid residue, (iv) RM is a reporter moiety. - View Dependent Claims (18, 19, 20, 21, 22)
- Y—
-
17. A fusion polypeptide, comprising segments P1 and Nux, wherein
(i) Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain, and (ii) P1 is a ligand binding polypeptide that binds to a non-peptide ligand of a hybrid ligand, which has the general formula R1— - Y—
R2, where R1 and R2 are ligands, R1 is different from R2, and at least one of R1 and R2 is not a peptide, and Y is a linker.
- Y—
-
23. A composition, comprising:
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(i) a hybrid ligand of the general formula R1—
Y—
R2, where R1 and R2 are ligands, R1 is different from R2 and at least one of R1 and R2 is not a peptide, Y is a linker; and
,(ii) at least one of two fusion polypeptides comprising;
(a) a first fusion polypeptide comprising segments P2, Cub-Z, and RM, in an order wherein Cub-Z is closer to the N-terminus of the first fusion polypeptide than RM, wherein P2 is a ligand binding polypeptide that may bind to ligand R1 or R2 of the hybrid ligand, Cub is a carboxy-terminal subdomain of ubiquitin and RM is a reporter moiety, and Z is an amino acid residue;
(b) a second fusion polypeptide comprising segments Nux and P1, wherein Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain, and P1 is a ligand binding polypeptide that may bind to ligand R1 or R2 of the hybrid ligand. - View Dependent Claims (26, 27)
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24. A composition, comprising:
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(i) a hybrid ligand represented by the general formula;
R1—
Y—
R2, wherein;
(a) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, 2,4-diaminopteridine derivative, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, or cyclosporin, or a derivative thereof with minor structural modifications;
(b) Y represents a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25;
(c) R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide;
(ii) at least one fusion polypeptide selected from;
(a) a first fusion polypeptide comprising;
a ligand binding domain P1 and a domain selected from the group consisting of;
a DNA binding domain and a transcriptional activation domain, wherein the ligand binding domain binds the first ligand R1; and
,(b) a second fusion polypeptide comprising;
a candidate ligand-binding domain P2 for the user-specified ligand R2 and a domain selected from the group consisting of;
a DNA binding domain and a transcriptional activation domain.wherein one of the first and second fusion polypeptides contains a DNA binding domain and the other fusion polypeptide contains a transcription activation domain;
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25. A composition comprising:
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(i) A hybrid ligand represented by the general formula;
R1—
Y—
R2, wherein;
(a) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, 2,4-diaminopteridine derivative, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, or cyclosporin or a derivative thereof with minor structural modifications;
(b) Y represents a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25;
(c) R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide; and
(ii) a fusion polypeptide that includes;
(a) at least one ligand binding domain; and
,(b) a functional domain heterologous to the ligand binding domain which by itself is not capable of inducing or allowing the detection of a detectable event, but which is capable of inducing or allowing the detection of a detectable event when brought into proximity of a second functional domain.
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28. A method of identifying a polypeptide sequence that binds to a user-specified ligand comprising:
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(i) providing a hybrid ligand having the general formula R1—
Y—
R2, where R1 is a first ligand, R2 is a user-specified ligand, and Y is a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25;
(ii) introducing the hybrid ligand into a population of cells, each cell containing a hybrid ligand screening system including;
(a) a reporter gene operably linked to a transcriptional regulatory sequence, said regulatory sequence including a DNA sequence which binds to a DNA binding domain;
(b) a first chimeric gene encoding a first fusion polypeptide comprising;
a ligand binding domain P1 and a domain selected from a DNA binding domain or a transcriptional activation domain, wherein the ligand binding domain binds the first ligand R1; and
,(c) a second chimeric gene encoding a second fusion polypeptide comprising;
a candidate ligand-binding domain P2 for the user-specified ligand R2 and a domain selected from a DNA binding domain or a transcriptional activation domain;
wherein one of the two fusion polypeptides contains a DNA binding domain and the other fusion polypeptide contains a transcription activation domain;
(iii) allowing the hybrid ligand to bind the ligand binding domain of the first fusion polypeptide through the first ligand R1 and to contact the candidate ligand binding domain of the second fusion polypeptide through the user-specified ligand R2 such that, if R2 binds to the candidate ligand binding domain, an increase in the level of transcription of the reporter gene occurs;
(iv) identifying a positive ligand binding cell in which an increase in the level of transcription of the reporter gene has occurred; and
,(v) identifying the nucleic acid sequence of the second chimeric gene encoding the candidate ligand binding domain that binds to the user-specified ligand R2, thereby identifying a polypeptide sequence that binds to a user-specified ligand. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 50, 51, 52, 53, 54, 55, 64)
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38. A method of identifying a polypeptide sequence that binds to a user-specified ligand comprising:
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(i) providing a hybrid ligand having the general formula R1—
Y—
R2, where R1 is a first ligand, R2 is a user-specified ligand different from R1, at least one of R1 and R2 is not a peptide, Y is a linker, and wherein R1 binds to or inhibits a kinase;
(ii) introducing the hybrid ligand into a population of cells, each cell containing a hybrid ligand screening system including;
(a) a reporter gene operably linked to a transcriptional regulatory sequence, said regulatory sequence including a DNA sequence which binds to a DNA binding domain;
(b) a first chimeric gene encoding a first fusion polypeptide comprising;
a ligand binding domain and a domain selected from the DNA binding domain or a transcriptional activation domain, wherein the ligand binding domain binds the first ligand R1; and
,(c) a second chimeric gene encoding a second fusion polypeptide comprising;
a candidate ligand-binding domain for the user-specified ligand R2 and a domain selected from the DNA binding domain or the transcription activation domain;
wherein one of the two fusion polypeptides contains a DNA binding domain and the other fusion polypeptide contains a transcription activation domain;
(iii) allowing the hybrid ligand to bind the ligand binding domain of the first fusion polypeptide through the first ligand R1 and to contact the candidate ligand binding domain of the second fusion polypeptide through the user-specified ligand R2 such that, if R2 binds to the candidate ligand binding domain, an increase in the level of transcription of the reporter gene occurs;
(iv) identifying a positive ligand binding cell in which an increase in the level of transcription of the reporter gene has occurred; and
,(v) identifying the nucleic acid sequence of the second chimeric gene encoding the candidate ligand binding domain that binds to the user-specified ligand R2, thereby identifying a polypeptide sequence that binds to a user-specified ligand. - View Dependent Claims (39, 40, 41, 42)
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43. A method of determining whether a polypeptide P2 and a ligand R2 bind to each other comprising:
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(i) translationally providing a first ligand-binding polypeptide comprising segments P1, Cub-Z, and RM, in an order wherein Cub-Z is closer to the N-terminus of the first ligand-binding polypeptide than RM, and a second ligand-binding polypeptide comprising segments Nux and P2, wherein P1 and P2 are polypeptides, Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain, Cub is the carboxy-terminal subdomain of a wild-type ubiquitin, Z is an amino acid residue and RM is a reporter moiety;
(ii) providing a hybrid ligand represented by the general formula;
R1—
Y—
R2, wherein R1 is a first ligand that binds the first ligand-binding polypeptide at P1, R2 is a second ligand different from R1, at least one of R1 and R2 is not a peptide, and Y is a linker;
(iii) allowing the hybrid ligand to contact the first and second ligand-binding polypeptides;
(iv) detecting the degree of cleavage by a ubiquitin-specific protease (UBP) of the first ligand-binding polypeptide between Cub and Z, wherein an increase of cleavage is indicative of polypeptide P2—
ligand R2 binding. - View Dependent Claims (45)
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44. A method of determining whether a polypeptide P1 and a ligand R1 bind to each other comprising:
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(i) translationally providing a first ligand-binding polypeptide comprising segments P1, Cub-Z, and RM, in an order wherein Cub-Z is closer to the N-terminus of the first ligand-binding polypeptide than RM, and a second ligand-binding polypeptide comprising segments Nux and P2, wherein P1 and P2 are polypeptides, Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain, Cub is the carboxy-terminal subdomain of a wild-type ubiquitin, Z is an amino acid residue and RM is a reporter moiety;
(ii) providing a hybrid ligand represented by the general formula;
R1—
Y—
R2, wherein R1 is a first ligand, R2 is a second ligand different from R1 that binds the second ligand-binding polypeptide at P2, at least one of R1 and R2 is not a peptide, and Y is a linker;
(iii) allowing the hybrid ligand to contact the first and second ligand-binding polypeptides;
(iv) detecting the degree of cleavage by a ubiquitin-specific protease (UBP) of the first ligand-binding polypeptide between Cub and Z, wherein an increase of cleavage is indicative of protein P1—
ligand R1 binding.
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46. A method of inducing or allowing the detection of a biologically detectable event, comprising:
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(i) providing at least one cell comprising at least one nucleic acid sequence encoding a fusion polypeptide that includes;
(a) at least one ligand binding domain; and
,(b) a functional domain which by itself is not capable of inducing or allowing the detection of the detectable event;
(ii) providing a hybrid ligand of the general formula R1—
Y—
R2, wherein R1 is different from R2, at least one of R1 and R2 is not a peptide, R1 or R2 represents a ligand that binds to said ligand binding domain;
Y represents a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25; and
wherein the binding of said hybrid ligand to said ligand binding domain brings the first functional domain into proximity of a second functional domain, thereby inducing or allowing the detection of the detectable event; and
,(iii) exposing said at least one cell to an effective amount of said hybrid ligand to bring the first functional domain into proximity of a second functional domain;
thereby inducing or allowing the detection of the biologically detectable event.
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47. A method of identifying a ligand of a user-specified polypeptide, comprising:
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(i) providing at least one candidate hybrid ligand having the general formula R1—
Y—
R2, where R1 is a first ligand, R2 is a candidate ligand, and Y is a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25;
(ii) introducing the candidate hybrid ligand into at least one cell which contains a hybrid ligand screening system including;
(a) a reporter gene operably linked to a transcriptional regulatory sequence, said regulatory sequence including a DNA sequence which binds to a DNA binding domain;
(b) a first chimeric gene encoding a first fusion polypeptide comprising;
a ligand binding domain and a domain selected from;
a DNA binding domain or a transcriptional activation domain, wherein the ligand binding domain binds the first ligand R1; and
,(c) a second chimeric gene encoding a second fusion polypeptide comprising;
a user-specified ligand-binding domain for the candidate ligand R2 and a domain selected from;
a DNA binding domain or a transcription activation domain;
wherein one of the two fusion polypeptides contains a DNA binding domain and the other fusion polypeptide contains a transcription activation domain;
(iii) allowing the candidate hybrid ligand to bind the ligand binding domain of the first fusion polypeptide through the first ligand R1 and to contact the user-specified ligand binding domain of the second fusion polypeptide through the candidate ligand R2 such that, if the user-specified ligand binding domain binds to the candidate ligand R2, an increase in the level of transcription of the reporter gene occurs;
(iv) identifying the candidate hybrid ligand which causes an increase in the level of transcription of the reporter gene in the cell, thereby identifying the candidate ligand on the candidate hybrid ligand as a ligand for the user-specified polypeptide. - View Dependent Claims (66)
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48. A method to investigate the structure activity relationship of a ligand to a ligand binding domain comprising:
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(i) providing a hybrid ligand R1—
Y—
R2, wherein(a) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridine derivative or cyclosporin or a derivative thereof with minor structural modifications;
(b) Y represents a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25; and
,(c) R2 represents a user-specified second ligand which is different from R1 and is selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide;
(ii) providing cells comprising a fusion protein that includes;
(a) at least one ligand binding domain; and
,(b) a functional domain heterologous to the ligand binding domain which by itself is not capable of inducing or allowing the detection of a detectable event, but which is capable of inducing or allowing the detection of a detectable event when brought into proximity of a second functional domain;
wherein either a plurality of hybrid ligands comprising structural variants of said second ligand R2 is provided in step (i), or a plurality of fusion proteins comprising structural variants of said ligand binding domain is provided in step (ii);
(iii) exposing said cells comprising each fusion protein to an effective amount of each hybrid ligand such that the first functional domain may be brought into proximity of a second functional domain thereby inducing or allowing the detection of a detectable event;
(iv) measuring the presence, amount or activity of any detectable event so induced or allowed in step (iii), thereby investigating the structure activity relationship between said second ligand and the ligand binding domain. - View Dependent Claims (49)
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56. A method to identify a hybrid ligand having the general structure R1—
- Y—
R2 suitable for an in-vivo assay, wherein said assay involves;
(i) the use of a hybrid ligand, and (ii) of at least one fusion polypeptide that includes;
(a) at least one ligand binding domain P; and
,(b) a functional domain which by itself is not capable of inducing or allowing the detection of the detectable event;
and wherein said method involves the steps of;
(iii) synthesizing a plurality of hybrid ligands R1—
Y—
R2 differing by a plurality of different linkers Y, wherein R1 and R2 are different, and at least one of R1 and R2 is not a peptide; and
(iv) testing each hybrid ligand in said plurality of hybrid ligands individually for efficacy in inducing or allowing the detection of the detectable event; and
(v) selecting a hybrid ligand with a particular linker that possesses suitable efficacy in inducing or allowing the detection of the detectable event. - View Dependent Claims (57, 58)
- Y—
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59. A kit comprising:
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at least one polynucleotide including a DNA fragment linked to a coding sequence for a functional domain heterologous to the DNA fragment which by itself is not capable of inducing or allowing the detection of a detectable event, but which is capable of inducing or allowing the detection of a detectable event when brought into proximity of a second functional domain;
and further comprising instructions (i) to synthesize a hybrid ligand of general structure R1—
Y—
R2, and(ii) to clone a ligand binding domain into the polynucleotide, and (iii) to test the binding between the hybrid ligand and the ligand binding domain, wherein R2 is different from R1, one of R1 and R2 is a non-peptide ligand, and wherein one of R1 and R2 binds to or inhibits a kinase.
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60. A kit comprising
at least one polynucleotide including a DNA fragment linked to a coding sequence for a functional domain heterologous to the DNA fragment which by itself is not capable of inducing or allowing the detection of a detectable event, but which is capable of inducing or allowing the detection of a detectable event when brought into proximity of a second functional domain; and further comprising instructions (i) to synthesize a hybrid ligand of general structure R1—
Y—
R2, and(ii) to clone a ligand binding domain into the polynucleotide, and (iii) to test the binding between the hybrid ligand and the ligand binding domain, wherein R2 is different from R1, one of R1 and R2 is a non-peptide ligand, and wherein Y is of the general structure (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25.
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61. A kit comprising
at least one polynucleotide including a DNA fragment linked to a coding sequence for a functional domain heterologous to the DNA fragment which by itself is not capable of inducing or allowing the detection of a detectable event, but which is capable of inducing or allowing the detection of a detectable event when brought into proximity of a second functional domain; and further comprising instructions (i) to synthesize a hybrid ligand of general structure R1—
Y—
R2, and(ii) to clone a ligand binding domain into the polynucleotide, and (iii) to test the binding between the hybrid ligand and the ligand binding domain, wherein R2 is different from R1, one of R1 and R2 is a non-peptide ligand, and wherein the functional domain is a carboxy-terminal subdomain of ubiquitin or an amino-terminal subdomain of ubiquitin.
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62. A kit comprising:
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(i) a compound of general structure R1—
Y—
L, wherein Y is of the general structure (CH2—
X—
CH2)n and L is a chemical group that is easily substituted by a different chemical group, and(ii) instructions to use the compound for the synthesis of a hybrid ligand R1—
Y—
R2 where R1 is different from R2, and at least one of R1 and R2 is not a peptide.
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63. A method of doing business comprising:
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(i) the identification of polypeptides binding to a hybrid ligand of general formula R1—
Y—
R2, wherein Y is of the general structure (CH2—
X—
CH2)n, R1 is different from R2, and at least one of R1 and R2 is not a peptide, X═
O, S, SO or SO2, and wherein said polypeptides were previously not known to bind to such hybrid ligand, and(ii) providing access to data, nucleic acids or polypeptides obtained from such identification to another party for consideration.
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65. A method of doing business comprising:
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(i) the identification of at least one ligand binding to a user-specified polypeptide by using a plurality of hybrid ligands of general formula R1—
Y—
R2 differing in at least one of R1 and R2, wherein R1 and R2 are ligands, R1 is different from R2, at least one of R1 and R2 is not a peptide, Y is of the general structure (CH2—
X—
CH2)n, X═
O, S, SO or SO2, and wherein said ligands were previously not known to bind to such polypeptide, and(ii) providing access to data and ligands obtained from such identification to another party for consideration.
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Specification