Method for producing diverse libraries of encoded polypeptides

Abstract

tRNA analogues which comprise a tRNA, such as tRNA phe ; an amino acid moiety which acts as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer and, thus, is stably linked to the acceptor stem of the tRNA; and a reactive or activatible moiety near or within the anticodon stem loop of the tRNA that can medidate the covalent coupling of the tRNA analogue to mRNA. Also described are polypeptide-tRNA analogue-mRNA fusions; libraries of encoded polypeptides; methods of producing and screening the libraries; and target members and their uses.

What is claimed is:  
     
         1 . A tRNA analogue, comprising: 
 (a) a tRNA;    (b) an amino acid moiety which acts as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer; and    (c) a reactive or activatible moiety near or within the anticodon stem-loop that can mediate the stable coupling of the tRNA analogue to mRNA.    
     
     
         2 . The tRNA analogue of  claim 1 , wherein the tRNA analogue is a 3′-amino-3′-deoxyadenosine-substituted tRNA or puromycin-substituted tRNA and the amino acid moiety is any amino acid or the methoxytyrosine moiety of a puromycin-substituted tRNA.  
     
     
         3  The tRNA analogue of  claim 1 , wherein the tRNA is yeast tRNA phe .  
     
     
         4 . The tRNA analogue of  claim 3 , wherein the 3′ terminal nucleotide of the yeast tRNA phe  has been replaced by 3′-amino-3′-deoxyadenosine or puromycin.  
     
     
         5 . A tRNA analogue which is a tRNA in which the 3′ terminal nucleotide is replaced by 3′-amino-3′-deoxyadenosine and then linked to an amino acid moiety or replaced by puromycin and in which the anticodon loop comprises a reactive or activatible moiety that can mediate covalent coupling of the tRNA analogue to mRNA.  
     
     
         6 . The tRNA analogue of  claim 5 , wherein the tRNA is yeast tRNA phe .  
     
     
         7 . The tRNA analogue of  claim 5 , wherein the reactive or activatible moiety is a modified base near or within the tRNA stem loop.  
     
     
         8 . The tRNA analogue of  claim 7  wherein the reactive or activatible moiety is a naturally modified guanine base at position 37.  
     
     
         9 . A polypeptide-tRNA analogue-mRNA fusion, comprising: 
 (a) a polypeptide;    (b) a tRNA analogue comprising: 
 (i) a tRNA;  
 (ii) an amino acid moiety which can act as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer; and  
 (iii) a reactive or activatible moiety near or within the anticodon stem-loom that can mediate the stable coupling of the tRNA analogue to mRNA; and  
   (c) mRNA which encodes the polypeptide of (a),     wherein the tRNA analogue is: located between the polypeptide and the mRNA; linked to the polypeptide by a stable bond between the terminal amino acid residue of the polypeptide and the amino acid moiety and, linked to the mRNA by crosslinks between a reactive or activatible moiety of the tRNA analogue and the mRNA.    
     
     
         10 . The polypeptide-tRNA analogue-mRNA fusion of  claim 9 , wherein the tRNA analogue is a 3′-amino-3′-deoxyadenosine-substituted tRNA or puromycin-substituted tRNA and the amino acid moiety is any amino acid or the methoxytyrosine moiety of puromycin.  
     
     
         11 . The polypeptide-tRNA analogue-mRNA fusion of  claim 9 , wherein the tRNA is yeast tRNA phe .  
     
     
         12 . The fusion of  claim 11 , wherein the tRNA is yeast tRNA phe  in which the 3′ terminal nucleotide has been replaced by 3′-amino-3′-deoxyadenosine or puromycin.  
     
     
         13 . A polypeptide-tRNA analogue-mRNA fusion, comprising: 
 (a) a polypeptide;    (b) a tRNA analogue comprising: 
 (i) a tRNA;  
 (ii) an amino acid moiety which can act as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer; and  
 (iii) a reactive or activatible moiety near or within the anticodon stem-loop that can mediate the stable coupling of the tRNA analogue to mRNA; and  
   (c) mRNA which encodes the polypeptide of (a),     wherein the tRNA analogue is: located between the polypeptide and the mRNA; linked to the polypeptide by a stable bond between the terminal amino acid residue of the polypeptide and the amino acid moiety; and linked to the mRNA by the action of UV irradiation that produces a crosslink between a modified base near or within the tRNA stem loop and the mRNA.    
     
     
         14 . The fusion of  claim 13 , wherein the tRNA analogue is a 3′-amino-3′-deoxyadenosine-substituted tRNA or puromycin-substituted tRNA and the amino acid moiety is any amino acid or the methoxytyrosine moiety of puromycin-substituted tRNA.  
     
     
         15 . The fusion of  claim 13 , wherein the tRNA is yeast tRNA phe  in which the 3′ terminal nucleotide has been replaced by 3′-amino-3′-deoxyadenosine or puromycin.  
     
     
         16 . A diverse library of encoded polypeptides, wherein the encoded polypeptides comprise: 
 (a) a polypeptide;    (b) a tRNA analogue comprising: 
 (i) a tRNA;  
 (ii) an amino acid moiety which can act as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer; and  
 (iii) a reactive or activatible moiety near or within the anticodon stem-loop that can mediate the stable coupling of the tRNA analogue to mRNA; and  
   (c) mRNA which encodes the polypeptide of (a),    wherein the tRNA analogue is: located between the polypeptide and the mRNA; linked to the polypeptide by a stable bond between the terminal amino acid residue of the polypeptide and the amino acid moiety; and linked to the mRNA by crosslinks between a reactive or activatible moiety of the tRNA analogue and the mRNA.    
     
     
         17 . The diverse library of  claim 16 , wherein the tRNA analogue is a 3′-amino-3′-deoxyadenosine-substituted tRNA or puromycin-substituted tRNA and the amino acid moiety is any amino acid or the methoxytyrosine moiety of puromycin-substituted tRNA.  
     
     
         18 . The diverse library of  claim 17 , wherein the tRNA is yeast tRNA phe .  
     
     
         19 . The library of  claim 18 , wherein the 3′ terminal nucleotide of yeast tRNA phe  has been replaced by 3′-amino-3′-deoxyadenosine or puromycin.  
     
     
         20 . A method of producing a diverse library of encoded polypeptides, which comprises polypeptide-tRNA analogue-mRNA fusions, comprising the steps of: 
 (a) combining: 
 (i) mRNAs which encode polypeptides;  
 (ii) tRNA analogues, wherein each tRNA analogue comprises: 
 (a) a tRNA;  
 (b) an amino acid moiety which can act as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer; and  
 (c) a reactive or activatible moiety near or within the anticodon stem-loop that can mediate the stable coupling of the tRNA analogue to mRNA; and  
 
 (iii) an appropriate in vitro translation mixture, thereby producing a combination;  
   (b) maintaining the combination under conditions appropriate for translation of the mRNAs to produce the encoded polypeptides and formation of a stable amino acid-tRNA analogue bond between the terminal amino acid residue of a polypeptide produced and the amino acid moiety present in the tRNA, to form polypeptide-tRNA analogue fusions, thereby producing a mixture which contains stalled ribosomes that contain polypeptide-tRNA analogue fusions; and    (c) exposing the mixture which contains stalled ribosomes that contain polypeptide-tRNA analogue fusions to conditions which favor the crosslinking of the tRNA analogue and the mRNA which encodes the polypeptide of the polypeptide-tRNA analogue fusion,    whereby polypeptide-tRNA analogue-mRNA fusions are produced, thereby producing a diverse library of encoded polypeptides.    
     
     
         21 . The method of  claim 20 , wherein the tRNA analogue is a 3′-amino-3′-deoxyadenosine-substituted tRNA or puromycin-substituted tRNA and the amino acid moiety is any amino acid or the methoxytyrosine moiety of puromycin-substituted tRNA.  
     
     
         22 . The method of  claim 20 , wherein the tRNA is yeast tRNA phe .  
     
     
         23 . The method of  claim 20 , wherein the 3′ terminal nucleotide of yeast tRNA phe  has been replaced with 3′-amino-3′-deoxyadenosine or puromycin and the conditions which favor crosslinking include mild ultraviolet irradiation.  
     
     
         24 . A method of identifying members of a diverse library of encoded polypeptides which exhibit a desired activity, wherein members are polypeptide-tRNA analogue-mRNA fusions, comprising the steps of: 
 (a) producing a diverse library of encoded polypeptides which comprises polypeptide tRNA analogue-mRNA fusions by: 
 (i) combining: 
 (1) mRNAs which encode polypeptides;  
 (2) tRNA analogues, wherein each tRNA analogue comprises: 
 (a) a tRNA;  
 (b) an amino acid moiety which can act as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer; and  
 (c) a reactive or activatible moiety near or within the anticodon stem-loop that can mediate the covalent coupling of the tRNA analogue to mRNA; and  
 
 (3) an appropriate in vitro translation mixture, thereby producing a combination;  
 
 (ii) maintaining the combination under conditions appropriate for translation of the mRNAs to produce the encoded polypeptides and formation of a stable amino acid-tRNA analogue bond between the terminal amino acid residue of a polypeptide produced and the amino acid moiety present in the tRNA analogue, to form polypeptide-tRNA analogue fusions, thereby producing a mixture which contains stalled ribosomes that contain polypeptide-tRNA analogue fusions; and  
 (iii) exposing the mixture which contains stalled ribosomes that contain polypeptide-tRNA analogue fusions to conditions which favor the crosslinking the tRNA analogue and the mRNA which encodes the polypeptide of the polypeptide-tRNA analogue fusion,  
  whereby polypeptide-tRNA analogue-mRNA fusions are produced, thereby producing a diverse library of encoded polypeptides;  
   (b) enriching the diverse library of encoded polypeptides for members which exhibit a desired activity, thereby producing an enriched diverse library comprised of polypeptide-tRNA analogue-mRNA fusions;    (c) amplifying the enriched diverse library by: 
 (i) reverse transcribing the mRNA components of the fusions, thereby producing the corresponding cDNA;  
 (ii) amplifying and transcribing in vitro the corresponding cDNA, thereby producing a pool of amplified, enriched mRNA from the corresponding cDNA;  
 (iii) combining the pool of amplified, enriched mRNA with an appropriate in vitro translation mixture and tRNA analogues of (a)(i)(2), thereby producing a combination;  
 (iv) maintaining the combination under conditions appropriate for translation of the mRNA to produce the encoded polypeptides and formation of a stable amino acid-tRNA analogue bond between the terminal amino acid residue of a polypeptide produced and the amino acid moiety present in the tRNA analogue, to form polypeptide-tRNA analogue fusions, thereby producing an amplified enriched mixture which contains stalled ribosomes that contain polypeptide-tRNA analogue fusion; and  
 (v) exposing the amplified enriched mixture which contains stalled ribosomes that contain polypeptide-tRNA analogue fusions to conditions which favor crosslinking of the tRNA analogue and the mRNA which encodes the polypeptide of the polypeptide-tRNA analogue fusion;  
   (d) repeating steps (b)-(c) as necessary until members which exhibit the desired activity are present in sufficient number to be detected; and    (e) detecting members which exhibit the desired activity, thereby identifying members which exhibit the desired activity.    
     
     
         25 . The method of  claim 24 , wherein the polypeptide-tRNA analogue-mRNA fusion is a 3′-amino-3′-deoxyadenosine-substituted tRNA or puromycin-substituted tRNA and the amino acid moiety is any amino acid or the methoxytyrosine moiety of puromycin.  
     
     
         26 . The method of  claim 25 , wherein in the polypeptide-tRNA analogue-mRNA fusion, the tRNA is yeast tRNA phe .  
     
     
         27 . The method of  claim 26 , wherein in the yeast tRNA phe , the 3′ terminal nucleotide has been replaced by 3′-amino-3′-deoxyadenosine or puromycin.  
     
     
         28 . A member of a diverse library of encoded polypeptides which exhibits a desired activity, identified by the method of  claim 24 .  
     
     
         29 . A polypeptide fragment of a member of a diverse library of encoded polypeptides, wherein the member exhibits a desired activity and is identified by the method of  claim 24 .  
     
     
         30 . A tRNA analogue-mRNA fragment of a member of a diverse library of encoded polypeptides, wherein the member exhibits a desired activity and is identified by the method of  claim 24.