Evolving new molecular function

• US 7,070,928 B2
• Filed: 03/19/2002
• Issued: 07/04/2006
• Est. Priority Date: 03/19/2001
• Status: Expired due to Term

• 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
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US10/101,030
• Publication Number:
  US 20030113738A1
• Time in Patent Office: 1,568 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 530/350
• US Class Current: 435/6.16
• 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. An in vitro method of performing synthesis from a nucleic acid template to produce a reaction product, wherein the reaction product is not a nucleic acid, the method comprising the steps of:

(a) providing (i) a template comprising a first reactive unit covalently attached to a first oligonucleotide defining a first codon sequence, and (ii) a transfer unit comprising a second reactive unit associated with a second oligonucleotide defining a first anti-codon sequence complementary to the first codon sequence of the template;

(b) annealing the first codon and first anti-codon sequences to bring the first reactive unit and the second reactive unit into reactive proximity; and

(c) inducing a reaction between the first and second reactive units without assistance of a ribosome to produce the reaction product.

2. The method of claim 1, wherein the second reactive unit is covalently attached to the second oligonucleotide.

3. The method of claim 1, wherein the template further comprises a second, different codon sequence.

4. The method of claim 3, further comprising providing a second transfer unit that anneals to the second, different codon sequence of the template.

5. The method of claim 4, wherein the first and second transfer units are provided together in step (b).

6. The method of claim 1, further comprising the additional step of selecting reaction product associated with the template.

7. The method of claim 6, wherein the reaction product is covalently attached to the template.

8. The method of claim 6, further comprising the additional step of amplifying the template.

9. The method of claim 6, further comprising the additional step of determining the sequence of the template thereby to facilitate identification of the reaction product.

55. The method of claim 1, wherein at least one of the reactive units is attached adjacent a terminal region of its corresponding oligonucleotide.

56. The method of claim 1, wherein each of the reactive units is attached adjacent a terminal region of its corresponding oligonucleotide.

57. The method of claim 1, wherein in the template the codon is disposed at least 10 bases away from its reactive unit.

58. The method of claim 1, wherein in the template the codon is disposed at least 20 bases away from its reactive unit.

59. The method of claim 1, wherein one of the oligonucleotides further comprise a pair of complementary sequences that anneal with one another to produce a hairpin.

60. The method of claim 1, wherein the template provided in step (a) further comprises an oligonucleotide sequence that identifies the first reactive unit.

61. The method of claim 1, wherein the reactive unit of the transfer unit is linked via a linker to the second oligonucleotide.

62. The method of claim 1, wherein the reaction product reaction remains attached by a linker to the second oligonucleotide of the transfer unit.

63. The method of claim 61, wherein the transfer unit comprises a linker that when cleaved leaves no atoms on the reaction product.

64. The method of claim 61, wherein the transfer unit comprises a linker that when cleaved leaves an atom on the reaction product.

65. The method of claim 61, wherein the reaction product is automatically cleaved from the second oligonucleotide during or after formation of the reaction product.

66. The method of claim 1, wherein the template is DNA or RNA.

67. The method of claim 1, wherein at least one of the template and the transfer unit is immobilized on a surface.

68. The method of claim 1, wherein the first reactive unit and the second reactive unit react spontaneously upon annealing of the oligonucleotides.

69. The method of claim 1, wherein the first reactive unit and the second reactive unit react upon exposure to a catalyst.

70. The method of claim 1, wherein the first reactive unit and the second reactive unit react in an aqueous solvent to produce a reaction product.

71. The method of claim 1, wherein the first reactive unit and the second reactive unit react in an organic solvent to produce a reaction product.

10. An in vitro method of performing synthesis from a nucleic acid template to produce a reaction product, wherein the reaction product is not a nucleic acid, the method comprising the steps of:

(a) providing (i) an amplifiable template comprising a first reactive unit associated with a first oligonucleotide defining a first codon sequence, and (ii) a first transfer unit comprising a second reactive unit associated with a second oligonucleotide defining an anti-codon sequence complementary to the first codon sequence;

(b) annealing the codon and anti-codon sequences to bring the first reactive unit and the second reactive unit into reactive proximity;

(c) inducing a reaction between the first and second reactive units without assistance of a ribosome to produce the reaction product; and

(d) amplifying the template that encoded the reaction product.

11. The method of claim 10, further comprising the additional step of determining the sequence of the template thereby to facilitate identification of the reaction product.

12. The method of claim 10, wherein in step (a), the template further comprises a second, different codon sequence.

13. The method of claim 12, wherein step (a) further comprises providing a second transfer unit that anneals to the second, different codon sequence of the template.

14. The method of claim 13, wherein the first and second transfer units are provided together in step (b).

15. The method of claim 10, wherein the first reactive unit is covalently attached to the first oligonucleotide.

16. The method of claim 10, wherein the first reactive unit is non-covalently attached to the first oligonucleotide.

17. The method of claim 16, wherein the first reactive unit is attached to the first oligonucleotide via a third oligonucleotide that hybridizes to the first oligonucleotide.

18. The method of claim 10, wherein the second reactive unit is covalently attached to the second oligonucleotide.

19. The method of claim 10, wherein the template is amplified by a polymerase chain reaction.

20. The method of claim 10, further comprising the additional step of selecting reaction product associated with the template.

21. The method of claim 20, wherein the reaction product is covalently attached to the template.

22. The method of claim 20, wherein the reaction product is non-covalently attached to the template.

23. An in vitro method of performing synthesis from a nucleic acid template to produce a reaction product, wherein the reaction product is not a nucleic acid, the method comprising the steps of:

(a) providing (i) a template comprising a first reactive unit associated with a first oligonucleotide defining a first codon sequence and a second, different codon sequence, and (ii) a first transfer unit comprising a second reactive unit associated with a second oligonucleotide defining a first anti-codon sequence complementary to the first codon sequence of the template;

(b) annealing the first codon sequence and the first anti-codon sequence to bring the first reactive unit and the second reactive unit into reactive proximity;

(c) inducing a reaction between the first and second reactive units without assistance of a ribosome to produce the reaction product; and

(d) selecting the reaction product still associated with the template.

24. The method of claim 23, further comprising providing a second transfer unit comprising a third oligonucleotide comprising a second anti-codon sequence that anneals to the second, different codon sequence of the template.

25. The method of claim 24, wherein the second transfer unit further comprises a third reactive unit.

26. The method of claim 25, further comprising, prior to step (d), the steps of:

(e) annealing the second anti-codon sequence to the second, different codon sequence of the template; and

(f) inducing a reaction between the third reactive unit and the reaction product, thereby to produce a modified reaction product to be selected in step (d).

27. The method of claim 26, wherein step (e) precedes step (c).

28. The method of claim 23, wherein the first reactive unit is covalently attached to the first oligonucleotide.

29. The method of claim 23, wherein the first reactive unit is non-covalently attached to the first oligonucleotide.

30. The method of claim 29, wherein the first reactive unit is non-covalently attached to the first oligonucleotide via an oligonucleotide sequence that hybridizes to the first oligonucleotide.

31. The method of claim 23, wherein the second reactive unit is covalently attached to the second oligonucleotide.

32. The method of claim 23, wherein the reaction product is covalently attached to the template.

33. The method of claim 23, further comprising the additional step of amplifying the template.

34. The method of claim 23, further comprising the additional step of determining the sequence of the template thereby to facilitate identification of the reaction product.

35. The method of claim 23, wherein the first reactive unit comprises a scaffold molecule that reacts with a plurality of reactants.

36. An in vitro method of performing synthesis from a nucleic acid template to produce a modified scaffold molecule, the method comprising the steps of:

(a) providing (i) a template comprising a scaffold molecule associated with a first oligonucleotide defining a first codon sequence and a second, different codon sequence, the scaffold molecule comprising a plurality of sites for modification, and (ii) a first transfer unit comprising a reactive unit covalently attached to a second oligonucleotide defining a first anti-codon sequence complementary to the first codon sequence;

(b) annealing the first codon sequence and the first anti-codon sequence to bring the scaffold molecule and the reactive unit into reactive proximity; and

(c) inducing a covalent bond-forming reaction between the reactive unit and the scaffold molecule without assistance of a ribosome to produce the modified scaffold molecule.

37. The method of claim 36, wherein the scaffold molecule is covalently attached to the first oligonucleotide.

38. The method of claim 36, wherein the reactive unit is covalently attached to the second oligonucleotide.

39. The method of claim 36, further comprising providing a second transfer unit comprising a reactive unit associated with a third oligonucleotide defining a second anti-codon sequence that anneals to the second, different codon sequence of the template.

40. The method of claim 36, further comprising the step of annealing the second anti-codon sequence of the second transfer unit to the second, different codon sequence of the template.

41. The method of claim 40, further comprising the step of inducing a covalent bond between the modified scaffold molecule and the second reactive unit.

42. The method of claim 36, further comprising the additional step of selecting a modified scaffold molecule associated with the template.

43. The method of claim 42, wherein the modified scaffold molecule is covalently attached to the template.

44. The method of claim 42, further comprising the step of amplifying the template.

45. The method of claim 42, further comprising the step of determining the sequence of the template to facilitate identification of the modified scaffold molecule.

46. The method of claim 44, further comprising the step of determining the sequence of the template to facilitate identification of the modified scaffold molecule.

47. The method of claim 40, wherein the first and second transfer units are provided together in step (b).

48. The method of claim 36, wherein the scaffold molecule reacts with a plurality of reactants.

49. An in vitro method of producing a plurality of different reaction products in a single solution, the method comprising the steps of:

(a) providing a plurality of different 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;

(b) providing a plurality of transfer units comprising a plurality of second reactive units covalently attached to a corresponding plurality of second oligonucleotides, wherein the second oligonueleotides each define an anti-codon sequence complementary to a codon sequence;

(c) annealing oligonucleotides having complementary codon and anti-codon sequences to bring the first and second reactive units into reactive proximity; and

(d) inducing covalent bond-forming reactions between the reactive units without the assistance of a ribosome to produce the plurality of different reaction products.

50. The method of claim 49, further comprising the step of selecting a reaction product having a desired property.

51. The method of claim 50, wherein at least one reaction product binds to a target molecule.

52. The method of claim 51, wherein the target molecule is a protein.

53. The method of claim 51, wherein the target molecule is immobilized on a solid support.

54. The method of claim 49, wherein the first reactive units associated with each template are different.