Evolving new molecular function

• US 20050227281A1
• Filed: 05/31/2005
• Published: 10/13/2005
• Est. Priority Date: 03/19/2001
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Liu, David R., Kanan, Matthew W., Gartner, Zev
• Granted Patent: US 7,442,160 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12P 1/00 Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00

Abstract

Nature evolves biological molecules such as proteins through iterated rounds of diversification, selection, and amplification. The present invention provides methods, compositions, and systems for synthesizing, selecting, amplifying, and evolving non-natural molecules based on nucleic acid templates. The sequence of a nucleic acid template is used to direct the synthesis of non-natural molecules such as unnatural polymers and small molecules. Using this method combinatorial libraries of these molecules can be prepared and screened. Upon selection of a molecule, its encoding nucleic acid template may be amplified and/or evolved to yield the same molecule or related molecules for re-screening. The inventive methods and compositions of the present invention allow for the amplification and evolution of non-natural molecules in a manner analogous to the amplification of natural biopolymer such as polynucleotides and protein.

Claims

1-46. -46. (canceled)

47. a method for selecting small molecules having binding affinity to a target molecule, the method comprising:

(a) mixing a target molecule and a library comprising small molecules associated with corresponding oligonucleotides, wherein an oligonucleotide has a nucleotide sequence informative of the synthetic history of the small molecule associated therewith, under conditions to permit the small molecules capable of binding the target molecule to bind thereto;

(b) separating small molecules that bind to the target molecule from unbound small molecules; and

(c) identifying the oligonucleotide associated with a small molecule that binds to the target molecule as indicative of binding affinity of the small molecule to the target molecule.

48. The method of claim 47 wherein step (c) comprises determining the sequence of the oligonucleotide associated with a small molecule that binds to the target molecule.

49. The method of claim 47 wherein the oligonucleotides comprise a primer pair to permit amplification of the oligonucleotides associated with small molecules that bind to the target molecule.

50. The method of claim 49 further comprising, after step (b) but before step (c), the step of amplifying the oligonucleotides associated with the separated small molecules.

51. The method of claim 47 wherein the small molecules comprise a plurality of reaction products prepared by a method comprising:

(i) providing a plurality of templates comprising a plurality of first reactive units associated with a corresponding plurality of first oligonucleotides, wherein each first oligonucleotide defines at least one codon sequence;

(ii) providing a plurality of transfer units comprising a plurality of second reactive units covalently attached to a corresponding plurality of second oligonucleotides, wherein each second oligonucleotide defines an anti-codon sequence complementary to a codon sequence; and

(iii) annealing oligonucleotides having complementary codon and anti-codon sequences to bring the first and second reactive units into reactive proximity thereby producing the plurality of reaction products.

52. The method of claim 47 wherein the nucleotide sequence encodes the synthesis of the small molecule associated therewith.

53. The method of claim 47 wherein the target molecule is a protein.

54. The method of claim 51 further comprising:

(iv) providing a plurality of second transfer units comprising a plurality of third reactive units covalently attached to a corresponding plurality of third oligonucleotides, wherein each third oligonucleotide defines an anti-codon sequence complementary to a second, different codon sequence; and

(v) annealing oligonucleotides having complementary codon and anti-codon sequences to bring the reaction products and third reactive units into reactive proximity thereby producing a plurality of second reaction products.

55. The method of claim 54 further comprising:

(vi) providing a plurality of third transfer units comprising a plurality of fourth reactive units covalently attached to a corresponding plurality of fourth oligonucleotides, wherein each fourth oligonucleotide defines an anti-codon sequence complementary to a third, different codon sequence; and

(vii) annealing oligonucleotides having complementary codon and anti-codon sequences to bring the second reaction products and fourth reactive units into reactive proximity thereby producing a plurality of third reaction products.

56. A method for selecting small molecules having binding affinity to a target molecule, the method comprising:

(a) mixing a target molecule and a library that comprises small molecules associated with corresponding oligonucleotides, wherein an oligonucleotide has a nucleotide sequence informative of the synthetic history of the small molecule associated therewith, under conditions to permit the small molecules capable of binding the target molecule to bind thereto;

(b) separating small molecules that bind to the target molecule from unbound small molecules;

(c) mutating the sequences of the oligonucleotides associated with the separated small molecules; and

(d) using the mutated sequences to direct synthesis of a new library of small molecules.

57. The method of claim 56 wherein, in step (c), the oligonucleotides are mutated by error-prone PCR.

58. The method of claim 56 wherein, in step (c), the oligonucleotides are mutated by in vitro homologous recombination.

59. A method for selecting small molecules having binding affinity to a target molecule, the method comprising:

(a) mixing a target molecule and a library that comprises small molecules associated with corresponding oligonucleotides, wherein an oligonucleotide has a nucleotide sequence informative of the structure of the small molecule associated therewith, under conditions to permit the small molecules capable of binding the target molecule to bind thereto;

(b) separating small molecules that bind to the target molecule from unbound small molecules; and

(c) identifying the oligonucleotide associated with a small molecule that binds to the target molecule as indicative of binding affinity of the small molecule to the target molecule.

60. The method of claim 59 wherein step (c) comprises determining the sequence of the oligonucleotide associated with a small molecule that binds to the target molecule.

61. The method of claim 59 wherein the oligonucleotides comprise a primer pair to permit amplification of the oligonucleotides associated with small molecules that bind to the target molecule.

62. The method of claim 61 further comprising, after step (b) but before step (c), the step of amplifying the oligonucleotides associated with the separated small molecules.

63. The method of claim 59 wherein the small molecules comprise a plurality of reaction products prepared by a method comprising:

(i) providing a plurality of templates comprising a plurality of first reactive units associated with a corresponding plurality of first oligonucleotides, wherein each first oligonucleotide defines at least one codon sequence;

(ii) providing a plurality of transfer units comprising a plurality of second reactive units covalently attached to a corresponding plurality of second oligonucleotides, wherein each second oligonucleotide defines an anti-codon sequence complementary to a codon sequence; and

(iii) annealing oligonucleotides having complementary codon and anti-codon sequences to bring the first and second reactive units into reactive proximity thereby producing the plurality of reaction products.

64. The method of claim 59 wherein the nucleotide sequence encodes the synthesis of the small molecule associated therewith.

65. The method of claim 63 further comprising:

(iv) providing a plurality of second transfer units comprising a plurality of third reactive units covalently attached to a corresponding plurality of third oligonucleotides, wherein each third oligonucleotide defines an anti-codon sequence complementary to a second, different codon sequence; and

(v) annealing oligonucleotides having complementary codon and anti-codon sequences to bring the reaction products and third reactive units into reactive proximity thereby producing a plurality of second reaction products.

66. The method of claim 65 further comprising:

(vi) providing a plurality of third transfer units comprising a plurality of fourth reactive units covalently attached to a corresponding plurality of fourth oligonucleotides, wherein each fourth oligonucleotide defines an anti-codon sequence complementary to a third, different codon sequence; and

(vii) annealing oligonucleotides having complementary codon and anti-codon sequences to bring the second reaction products and fourth reactive units into reactive proximity thereby producing a plurality of third reaction products.

67. A method for selecting small molecules having binding affinity to a target molecule, the method comprising:

(a) mixing a target molecule and a library that comprises small molecules, wherein each small molecule is associated with corresponding oligonucleotides, and wherein an oligonucleotide has a nucleotide sequence informative of the structure of the small molecule associated therewith, under conditions to permit the small molecules capable of binding the target molecule to bind thereto;

(b) separating small molecules that bind to the target molecule from unbound small molecules;

(c) mutating the sequences of the oligonucleotides associated with the separated small molecules; and

(d) using the mutated sequences to direct synthesis of a new library of small molecules.

68. The method of claim 67 wherein, in step (c), the oligonucleotides are mutated by error-prone PCR.

69. The method of claim 67 wherein, in step (c), the oligonucleotides are mutated by in vitro homologous recombination.

70. The method of claim 67 wherein the target molecule is a protein.

71. A method for evolving a library of chemical compounds, the method comprising:

(a) providing a library of chemical compounds, wherein the chemical compounds are associated with corresponding oligonucleotides, and wherein an oligonucleotide has a nucleotide sequence informative of the synthetic history of the chemical compound associated therewith;

(b) selecting the oligonucleotides associated with the chemical compounds having a desired property;

(c) mutating the sequences of the selected oligonucleotides; and

(d) synthesizing a new library of chemical compounds using the mutated sequences.

72. The method of claim 71 wherein a desired property is selected from the group consisting of catalytic activity and binding activity.

73. The method of claim 71 wherein the desired property is binding activity to a target molecule.

74. The method of claim 73 wherein the target molecule is a protein.

75. The method of claim 71 wherein, in step (c), the sequence of the oligonucleotides are mutated by error-prone PCR.

76. The method of claim 71 wherein, in step (c), the sequence of the oligonucleotides are mutated by in vitro homologous recombination.

77. A method for evolving a library of chemical compounds, the method comprising:

(a) providing a library of chemical compounds, the chemical compounds being associated with corresponding oligonucleotides, wherein an oligonucleotide has a nucleotide sequence informative of the structure of the chemical compound associated therewith;

(b) selecting the oligonucleotides associated with the chemical compounds having a desired property;

(c) mutating the sequences of the selected oligonucleotides; and

(d) synthesizing a new library of chemical compounds using the mutated sequences.

78. The method of claim 77 wherein the desired property is selected from the group consisting of catalytic activity and binding activity.

79. The method of claim 77 wherein the desired property is binding activity to a target molecule.

80. The method of claim 79 wherein the target molecule is a protein.

81. The method of claim 77 wherein, in step (c), the sequence of the oligonucleotides are mutated by error-prone PCR.

82. The method of claim 77 wherein, in step (c), the sequence of the oligonucleotides are mutated by in vitro homologous recombination.