Novel methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries

Abstract

Methods useful in constructing libraries that collectively display and/or express members of diverse families of peptides, polypeptides or proteins and the libraries produced using those methods. Methods of screening those libraries and the peptides, polypeptides or proteins identified by such screens.

1 .- 116 . (canceled) 
     
     
         117 . A method for preparing single-stranded nucleic acids, the method comprising the steps of:
 (i) contacting a single-stranded nucleic acid sequence that has been cleaved with a restriction endonuclease with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acids in the region that remains after cleavage, the double-stranded region of the oligonucleotide including any sequences necessary to return the sequences that remain after cleavage into proper and original reading frame for expression and containing a restriction endonuclease recognition site 5′ of those sequences; and   (ii) cleaving the partially double-stranded oligonucleotide sequence solely at the restriction endonuclease recognition site contained within the double-stranded region of the partially double-stranded oligonucleotide,   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.   
     
     
         118 . The method according to  claim 117 , wherein the length of the single-stranded portion of the partially double-stranded oligonucleotide is between 2 and 15 bases. 
     
     
         119 . The method according to  claim 118 , wherein the length of the single-stranded portion of the partially double-stranded oligonucleotide is between 7 and 10 bases. 
     
     
         120 . The method according to  claim 117 , wherein the length of the double-stranded portion of the partially double-stranded oligonucleotide is between 12 and 100 base pairs. 
     
     
         121 . The method according to  claim 120 , wherein the length of the double-stranded portion of the partially double-stranded oligonucleotide is between 20 and 100 base pairs. 
     
     
         122 . A method for preparing a library comprising a collection of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and that collectively display at least a portion of the family comprising the steps:
 (i) preparing a collection of nucleic acids that code at least in part for members of the diverse family;   (ii) rendering the nucleic acids single-stranded;   (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of:
 (a) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in, the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and 
 (b) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide; 
   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature;   (iv) contacting the nucleic acid with a partially double-stranded oligonucleotide, the singles stranded region of the oligonucleotide being functionally complementary to the nucleic acids in the region that remains after the cleavage in step (iii) has been effected, and the double-stranded region of the oligonucleotide including any sequences necessary to return the sequences that remain after cleavage into proper and original reading frame for display and containing a restriction endonuclease recognition site 1 of those sequences that is different from the restriction site used in step (iii); and   (v) cleaving the nucleic acid solely at the restriction endonuclease recognition cleavage site contained within the, double-stranded region of the partially double-stranded oligonucleotide;   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a cleavage endonuclease that is active at the chosen temperature; and   (vi) displaying a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids on the surface of the genetic package and collectively displaying at least a portion of the diversity of the family.   
     
     
         123 . A method for preparing a library comprising a collection of members of a diverse family of peptides, polypeptides or proteins and collectively comprising at least a portion of the family comprising the steps:
 (i) preparing a collection of nucleic acids that code at least in part for members of the diverse family;   (ii) rendering the nucleic acids single-stranded;   (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of:
 (a) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and 
 (b) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide; 
   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature;   (iv) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acids in the region that remains after the cleavage in step (iii) has been effected, and the double-stranded region of the oligonucleotide including any sequence necessary to return the sequences that remain after cleavage into proper and original reading frame for expression and containing a restriction endonuclease recognition site 5′ of those sequences that is different from the restriction site used in step (iii); and   (v) cleaving the nucleic acid solely at the restriction endonuclease recognition cleavage site contained within the double-stranded region of the partially double-stranded oligonucleotide;   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a cleavage endonuclease that is active at the chosen temperature; and   (vi) expressing a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids and collectively expressing at least a portion of the diversity of the family.   
     
     
         124 . The method according to  claim 122 , further comprising at least one nucleic acid amplification step between one or more of steps (i) and (iii) steps (ii) and (iii), steps (iii) and (iv) and steps (iv) and (v). 
     
     
         125 . A library comprising a collection of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and collectively display at least a portion of the diversity of the family, the library being produced using the method of  claim 122 . 
     
     
         126 . A library comprising a collection of members of a diverse family of peptides, polypeptides or proteins and collectively comprise at least a portion of the diversity of the family, the library being produced using the method of  claim 123 . 
     
     
         127 . The method or library according to  claim 122 , wherein the members of the library encode an immunoglobulin. 
     
     
         128 . The method or library according to  claim 127 , wherein the double-stranded region of the oligonucleotide encodes at least a part of a framework sequence of an immunoglobulin. 
     
     
         129 . The method or library according to  claim 128 , wherein the framework sequence comprises framework 1 of an antibody. 
     
     
         130 . The method or library according to  claim 129 , wherein the framework sequence comprises framework 1 of a variable domain of a light chain. 
     
     
         131 . The method or library according to  claim 129 , wherein the framework sequence comprises framework 1 of a variable domain of a heavy chain. 
     
     
         132 . A method for displaying a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a portion of the diversity of the family, the method comprising the steps of:
 (i) preparing a collection of nucleic acids that code, at least in part, for members of the diverse family;   (ii) rendering the nucleic acids single-stranded;   (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of:
 (a) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid at its 5′ terminal region; and 
 (b) cleaving the nucleic acid solely at a restriction endonuclease cleavage site located in the double-stranded region of the oligonucleotide or amplifying the nucleic acid using a primer at least in part functionally complementary to at least a part of the double-stranded region of the oligonucleotide, the primer also introducing on amplification an endonuclease cleavage site and cleaving the amplified nucleic acid sequence solely at that site; 
   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a cleavage endonuclease that is active at the chosen temperature; and   (iv) displaying a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids on the surface of the genetic package and collectively displaying at least a portion of the diversity of the family.   
     
     
         133 . A method for expressing a member of a diverse family of peptides, polypeptides or proteins and collectively expressing at least a portion of the diversity of the family, the method comprising the steps of:
 (i) preparing a collection of nucleic acids that code, at least in part, for members of the diverse family;   (ii) rendering the nucleic acids single-stranded;   (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of:
 (a) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid at its 5′ terminal region; and 
 (b) cleaving the nucleic acid solely at a restriction endonuclease cleavage site located in the double-stranded region of the nucleotide; or amplifying the nucleic acid using a primer at least in part functionally complementary to at least a part of the double-stranded region of the oligonucleotide, the primer also introducing on amplification an endonuclease cleavage site and cleaving the amplified nucleic acid sequence solely at that site; 
   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a cleavage endonuclease that is active at the chosen temperature; and (iv) expressing a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids and collectively expressing at least a portion of the diversity of the family.   
     
     
         134 . The method according to  claim 132 , further comprising at least one nucleic acid amplification step between one or more of steps (i) and (ii), steps (ii) and (iii), and steps (iii) and (iv). 
     
     
         135 . A library comprising a collection of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and collectively display at least a portion of the diversity of the family, the library being produced using the method of  claim 132 . 
     
     
         136 . A library comprising a collection of members of a diverse family of peptides, polypeptides or proteins and collectively comprise at least a portion of the diversity of the family, the library being produced using the method of  claim 133 . 
     
     
         137 . The method according to  claim 132 , wherein the members of the library encode immunoglobulins. 
     
     
         138 . A method for cleaving a nucleic acid sequence at a desired location, the method comprising the steps of:
 (i) contacting a single-stranded nucleic acid sequence with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the 5′ terminal region of the nucleic acid sequence, the double-stranded region of the oligonucleotide including any sequences necessary to return the sequence in the single-stranded nucleic acid sequence into proper and original reading frame for expression; and   (ii) cleaving the partially double-stranded oligonucleotide-single-stranded nucleic acid combination solely at a restriction endonuclease cleavage site contained within the double-stranded oligonucleotide or amplifying the combination using a primer at least in part functionally complementary to at least part of the double-stranded region of the oligonucleotide, the primer introducing during amplification an endonuclease cleavage site and cleaving the amplified sequence solely at the site.   
     
     
         139 . The method according to  claim 138 , wherein the length of the single-stranded portion of the partially double-stranded oligonucleotide is between 2 and 15 bases. 
     
     
         140 . The method according to  claim 139 , wherein the length of the single-stranded portion of the partially double-stranded oligonucleotide is between 7 and 10 bases. 
     
     
         141 . The method according to  claim 138 , wherein the length of the double-stranded portion of the partially double-stranded oligonucleotide is between 12 and 100 base pairs. 
     
     
         142 . The method according to  claim 141 , wherein the length of the double-stranded portion of the partially double-stranded oligonucleotide is between 20 and 100 base pairs. 
     
     
         143 . The method according to  claim 138 , further comprising at least one nucleic acid amplification step between steps (i) and (ii).