Evolving new molecular function

• US 20050142583A1
• Filed: 09/24/2004
• Published: 06/30/2005
• 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 7,223,545 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. An in vitro method of inducing a chemical reaction without the assistance of a ribosome, the method comprising the steps of:

(a) providing (i) a first reactive unit covalently attached to a first oligonucleotide, and (ii) a second reactive unit covalently attached to a second oligonucleotide capable of hybridizing to the first oligonucleotide; and

(b) annealing the first and second oligonucleotides to bring the first reactive unit and the second reactive unit into reactive proximity so that the reactive units, without the assistance of a ribosome, react with one another to produce a reaction product.

48. The method of claim 47, wherein the first oligonucleotide comprises a sequence that identifies the first reactive unit.

49. The method of claim 48, wherein the second oligonucleotide comprises a sequence that identifies the second reactive unit.

50. The method of claim 48, wherein the sequence that identifies the first reactive unit is a codon sequence.

51. The method of claim 48, wherein the sequence that identifies the first reactive unit is an anti-codon sequence.

52. The method of claim 47, wherein the annealed first and second oligonucleotides are present in a complex comprising a sequence identifying the second reactive unit.

53. The method of claim 48 further comprising the step of determining the sequence of the sequence identifying the first reactive unit thereby to facilitate identification of the reaction product.

54. The method of claim 49 further comprising the step of determining the sequence identifying the second reactive unit thereby to facilitate identification of reaction product.

55. The method of claim 47 further comprising the step of selecting the reaction product.

56. An in vitro method of inducing a chemical reaction without the assistance of a ribosome, the method comprising the steps of:

(a) providing (i) a first reactive unit covalently attached to a first oligonucleotide, and (ii) a second reactive unit covalently attached to a second oligonucleotide capable of hybridizing to the first oligonucleotide and comprising a sequence indicative of the second reactive unit; and

(b) annealing the first and second oligonucleotides to bring the first reactive unit and the second reactive unit into reactive proximity so that the reactive units, without the assistance of a ribosome, react with one another to produce a reaction product.

57. The method of claim 56, wherein the first oligonucleotide comprises a sequence that identifies the first reactive unit.

58. The method of claim 56, wherein the sequence that identifies the second reactive unit is a codon sequence.

59. The method of claim 56, wherein the sequence that identifies the second reactive unit is an anti-codon sequence.

60. The method of claim 56, wherein the annealed first and second oligonucleotides are present in a complex comprising a sequence identifying the second reactive unit.

61. The method of claim 56 comprising the additional step of determining the sequence of the sequence identifying the second reactive unit thereby to facilitate identification of the reaction product.

62. The method of claim 56 comprising the additional step of selecting the reaction product.

63. An in vitro method of inducing a chemical reaction without the assistance of a ribosome, the method comprising the steps of:

(a) providing a first reactive unit, a first oligonucleotide comprising a sequence indicative of the identity of the first reactive unit, and a second oligonucleotide covalently attached to a small molecule scaffold comprising sites for functionalization; and

(b) forming a complex comprising the first and second oligonucleotides wherein the first reactive unit, without the assistance of a ribosome, reacts with the scaffold at one of the sites for functionalization to produce a reaction product, and wherein the complex comprises a sequence identifying the reactive unit that reacted with the scaffold.

64. The method of the claim 63, further comprising:

(c) providing a second reactive unit and a third oligonucleotide comprising a sequence indicative of the identity of the second reactive unit; and

(d) forming a complex comprising the second and third oligonucleotides such that the second reactive unit, without the assistance of a ribosome, reacts with the scaffold at a second one of the sites for functionalization.

65. The method of claim 64, wherein the first, second and third oligonucleotides are present in the same mixture.

66. The method of claim 64 or 65, wherein the first oligonucleotide in covalently attached to the first reactive unit.

67. The method of claim 65, wherein the third oligonucleotide is covalently attached to the second reactive unit.

68. The method of claim 63 comprising the additional step of determining the sequence of the sequence identifying the first reactive unit that reacted with the scaffold template thereby to facilitate identification of the reaction product.

69. The method of claim 63 comprising the additional step of determining the sequence of the sequence identifying the second reactive unit that reacted with the scaffold template thereby to facilitate identification of the reaction product.

70. The method of claim 63 comprising the additional step of selecting the reaction product.

71. A composition comprising:

(a) a small molecule scaffold covalently attached to an oligonucleotide, wherein the small molecule scaffold comprises one or more functionalization sites; and

(b) a building block provided by a reactive unit covalently attached to the small molecule scaffold via a functionalization site,

wherein the oligonucleotide comprises a sequence that identifies the building block covalently bonded to the scaffold via the functionalization site.

73. The composition of claim 71 or 72, wherein the oligonucleotide further comprises a pair of primer binding sequences to permit amplification of the oligonucleotide by polymerase chain reaction.

74. The composition of claim 71 or 72, wherein the oligonucleotide further comprises a sequence indicative of the identity of the small molecule scaffold.

72. A composition comprising:

(a) a small molecule scaffold covalently attached to an oligonucleotide; and

(b) a plurality of building blocks provided by reactive units covalently attached to the small molecule scaffold, wherein each building block is covalently bonded to a different site on the scaffold, and wherein the oligonucleotide comprises a plurality of sequences, each of which identifies a building block covalently attached to the scaffold.