Novel methods of constructing libraries of genetic packages that collectively display the members of a diverse family of peptides, polypeptides or proteins
First Claim
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1. A method for cleaving single-stranded nucleic acid sequences at a desired location, the method comprising the steps of:
- (i) 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
(ii) 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.
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Abstract
Methods useful in constructing libraries that collectively display 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.
137 Citations
82 Claims
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1. A method for cleaving single-stranded nucleic acid sequences at a desired location, the method comprising the steps of:
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(i) 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
(ii) 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. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 29, 42, 43, 44, 45, 46, 47)
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2. A method for cleaving single-stranded nucleic acid sequences at a desired location, the method comprising the steps of:
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(i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a Type II-S restriction endonuclease recognition site, whose cleavage site is located at a known distance from the recognition site; and
(ii) cleaving the nucleic acid solely at the Type II-S cleavage site formed by the complementation of the nucleic acid and the single-stranded region of 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. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36)
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3. In 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 part of the diversity of the family, the improvement being characterized in that the displayed at least a part of peptide, polypeptide or protein is encoded at least in part by a nucleic acid that has been cleaved at a desired location by a method comprising the steps of:
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(i) 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
(ii) 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.
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4. In 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 part of the diversity of the family, the improvement being characterized in that the displayed peptide, polypeptide or protein is encoded by a DNA sequence comprising a nucleic acid that has been cleaved at a desired location by
(i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a Type II-S restriction endonuclease recognition site, whose cleavage site is located at a known distance from the recognition site; - and
(ii) cleaving the nucleic acid solely at the Type I1-S cleavage site formed by the complementation of the nucleic acid and the single-stranded region of 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 desires location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
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5. 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 part of the diversity of the family, the method comprising the steps of:
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(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; 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. - View Dependent Claims (21, 22)
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6. 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:
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(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 in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a Type II-S restriction endonuclease recognition site, whose cleavage site is located at a known distance from the recognition site; and
(b) cleaving the nucleic acid solely at the Type II-S cleavage site formed by the complementation of the nucleic acid and the single-stranded region of 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 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.
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7-9. -9. (canceled)
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37. A method for preparing single-stranded nucleic acids for cloning into an vector, the method comprising the steps of:
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(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 acid 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. - View Dependent Claims (38, 39, 40, 41)
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48. A method for preparing single-stranded nucleic acids, the method comprising the steps of:
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(i) 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
(ii) 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.
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49. A method for preparing single-stranded nucleic acids, the method comprising the steps of:
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(i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a Type I1-S restriction endonuclease recognition site, whose cleavage site is located at a known distance from the recognition site; and
(ii) cleaving the nucleic acid solely at the cleavage site formed by the complementation of the nucleic acid and the single-stranded region of 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. - View Dependent Claims (50, 51, 52, 53)
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54. 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 diversity of the family comprising the steps:
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(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 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 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) 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. - View Dependent Claims (55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68)
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69. A method for producing a population of immunoglobulin genes, the method comprising:
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(i) introducing synthetic diversity into at least one of CDR1 or CDR2 of those genes; and
(ii) combining the diversity from step (i) with CDR3 diversity captured from B cells. - View Dependent Claims (70)
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71. A method for producing a library of immunoglobulin genes, the method comprising:
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(i) introducing synthetic diversity into at least one of CDR1 or CDR2 of those genes; and
(ii) combining the diversity from step (i) with CDR3 diversity captured from B cells. - View Dependent Claims (72)
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73. In 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 part of the diversity of the family, the improvement being characterized in that the displayed peptide, polypeptide or protein is encoded by a DNA sequence comprising a nucleic acid that has been cleaved at a desired location by
(i) 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 and
(ii) 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 cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
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74. 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:
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(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.
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75. 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 diversity of the family comprising the steps:
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(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 5′
terminal 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
(v) cleaving the nucleic acid solely at a restriction endonuclease cleavage site contained within the double-stranded region of the partially double-stranded oligonucleotide, the site being different horn that used in step (iii)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
(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. - View Dependent Claims (81, 82)
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76. A method for cleaving a nucleic acid sequence at a desired location, the method comprising the steps of:
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(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. - View Dependent Claims (77, 78, 79, 80)
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Specification