Evolving new molecular function

• US 7,491,494 B2
• Filed: 08/19/2003
• Issued: 02/17/2009
• Est. Priority Date: 08/19/2002
• Status: Expired due to Fees

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Liu, David R., Gartner, Zev J., Calderone, Christopher T.
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US10/643,752
• Publication Number:
  US 20040180412A1
• Time in Patent Office: 2,009 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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: C12Q 1/68 involving nucleic acids

Abstract

Nature evolves biological molecules such as proteins through iterated rounds of diversification, selection, and amplification. The power of Nature and the flexibility of organic synthesis are combined in nucleic acid-templated synthesis. The present invention provides a variety of template architectures for performing nucleic acid-templated synthesis, methods for increasing the selectivity of nucleic acid-templated reactions, methods for performing stereoselective nucleic acid-templated reactions, methods of selecting for reaction products resulting from nucleic acid-templated synthesis, and methods of identifying new chemical reactions based on nucleic acid-templated synthesis.

Claims

1. An in vitro method of increasing reaction selectivity among a plurality of reactants in a nucleic acid-templated synthesis to produce, without the assistance of a ribosome, a reaction product that is not a nucleic acid, the method comprising the steps of:

(a) providing (i) a template associated with a capturable moiety and comprising a first reactive unit associated with a first oligonucleotide comprising a predetermined codon sequence, (ii) a first transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon sequence capable of annealing to said codon sequence, and (iii) a second transfer unit comprising a third reactive unit different from said second reactive unit associated with a third oligonucleotide without an anti-codon sequence capable of annealing to said codon sequence, wherein said second reactive unit and said third reactive unit are capable of reacting independently with said first reactive unit and said second reactive unit and said third reactive unit are capable of reacting with one another; and

(b) mixing said template, said first transfer unit and said second transfer unit under conditions to permit annealing of said second oligonucleotide of said first transfer unit to said first oligonucleotide of said template thereby to enhance covalent bond formation between said second reactive unit and said first reactive unit to produce the reaction product relative to covalent bond formation between said third reactive unit and said first reactive unit.

2. The method of claim 1, wherein said first transfer unit is associated with a capturable moiety.

3. The method of claim 1, wherein said second transfer unit is associated with a capturable moiety.

4. The method of claim 1, 2, or 3, wherein said capturable moiety is selected from the group consisting of biotin, avidin and streptavidin.

5. The method of claim 4, further comprising the step of capturing said capturable moiety.

6. The method of claim 1 wherein said first reactive unit is covalently attached to said first oligonucleotide.

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

8. The method of claim 1, wherein said third reactive unit is covalently attached to said third oligonucleotide.

10. The method of claim 1, comprising providing a plurality of transfer units.

20. The method of claim 1, further comprising:

providing a second template comprising a fourth reactive unit associated with a fourth oligonucleotide comprising a second predetermined codon sequence, different from said predetermined codon sequence of said first oligonucleotide, wherein said second predetermined codon sequence is capable of annealing with said third oligonucleotide; and

mixing said second template with said first transfer unit, said second transfer unit, and said template comprising said first reactive unit associated with said first oligonucleotide under conditions to permit

annealing of said second oligonucleotide of said first transfer unit to said first oligonucleotide of said template and, in the same solution,

annealing of said third oligonucleotide of said second transfer unit to said fourth oligonucleotide of said second template, thereby to induce covalent bond formation both between said second reactive unit and said first reactive unit and between said fourth reactive unit and said third reactive unit.

9. The method of 1, wherein the reaction between said second reactive unit and said third reactive unit are incompatible with their respective reactions with said first reactive unit.

11. An in vitro method of increasing reaction selectivity among a plurality of reactants in a nucleic acid-templated synthesis to produce, without the assistance of a ribosome, a reaction product that is not a nucleic acid, the method comprising the steps of:

(a) providing (i) a template associated with a capturable moiety and comprising a first oligonucleotide comprising first and second codon sequences, (ii) a first transfer unit comprising a first reactive unit associated with a second oligonucleotide comprising a first anti-codon sequence capable of annealing to said first codon sequence, (iii) a second transfer unit comprising a second reactive unit associated with a third oligonucleotide comprising a second anti-codon sequence capable of annealing to said second codon sequence, and (iv) a third transfer unit comprising a third reactive unit associated with a fourth oligonucleotide sequence without an anti-codon sequence capable of annealing to said first codon sequence or said second codon sequence, wherein said third reactive unit is capable of reacting with said first reactive unit and said second reactive unit; and

p1 (b) mixing said template, said first transfer unit, said second transfer unit and said third transfer unit under conditions to permit annealing of said first anti-codon sequence to said first codon sequence and said second anti-codon sequence to said second codon sequence thereby to enhance covalent bond formation between said first reactive unit and said second reactive unit to produce the reaction product relative to covalent bond formation between said third reactive unit and said first reactive unit or between said third reactive unit and said second reactive unit.

12. The method of claim 11, wherein said capturable moiety is selected from the group consisting of biotin, avidin and streptavidin.

13. The method of claim 11, wherein said capturable moiety is a reaction product resulting from a reaction between said first reactive unit and said second reactive unit when said first transfer unit and said second transfer unit are annealed to said template.

14. The method of claim 11, wherein said first reactive unit is covalently attached to said second oligonucleotide.

15. The method of claim 11, wherein said second reactive unit is covalently attached to said third oligonucleotide.

16. The method of claim 11, wherein said third reactive unit is covalently attached to said fourth oligonucleotide.

17. The method of claim 11, wherein the reaction between said third reactive unit and said first reactive unit is incompatible with the reaction between said first reactive unit and said second reactive unit.

18. The method of claim 11, wherein the reaction between said third reactive unit and said second reactive unit is incompatible with the reaction between said first reactive unit and said second reactive unit.

19. The method of claim 11, wherein said covalent bond formation between said first reactive unit and said second reactive unit is via a regioselective distance dependent reaction.

21. An in vitro method of increasing reaction selectivity among a plurality of reactants in a nucleic acid-templated synthesis to produce, without the assistance of the ribosome, a reaction product that 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 comprising a predetermined codon sequence, (ii) a first transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon sequence capable of annealing to said codon sequence, and (iii) a second transfer unit comprising a third reactive unit different from said second reactive unit associated with a third oligonucleotide without an anti-codon sequence capable of annealing to said codon sequence; and

(b) mixing said template, said first transfer unit and said second transfer unit under conditions to permit annealing of said second oligonucleotide of said first transfer unit to said first oligonucleotide of said template thereby to enhance covalent bond formation between said second reactive unit and said first reactive unit to produce the reaction product relative to covalent bond formation between said third reactive unit and said first reactive unit,

wherein said second reactive unit and said third reactive unit are capable of reacting with one another and the reaction between said second reactive unit and said third reactive unit is incompatible with the reaction between said second reactive unit and said first reactive unit.

22. An in vitro method of increasing reaction selectivity among a plurality of reactants in a nucleic acid-templated synthesis to produce, without the assistance of the ribosome, a reaction product that is not a nucleic acid, the method comprising the steps of:

(a) providing (i) a template comprising a first oligonucleotide comprising first and second codon sequences, (ii) a first transfer unit comprising a first reactive unit associated with a second oligonucleotide comprising a first anti-codon sequence capable of annealing to said first codon sequence, (iii) a second transfer unit comprising a second reactive unit associated with a third oligonucleotide comprising a second anti-codon sequence capable of annealing to said second codon sequence, and (iv) a third transfer unit comprising a third reactive unit different from said second reactive unit associated with a fourth oligonucleotide sequence without an anti-codon sequence capable of annealing to said first codon sequence or said second codon sequence; and

(b) mixing said template, said first transfer unit, said second transfer unit and said third transfer unit under conditions to permit annealing of said first anti-codon sequence to said first codon sequence and said second anti-codon sequence to said second codon sequence thereby to enhance covalent bond formation between said first reactive unit and said second reactive unit to produce the reaction product relative to covalent bond formation between said third reactive unit and said first reactive unit or between said third reactive unit and said second reactive unit,

wherein said third reactive unit is capable of reacting with said second reactive unit and the reaction between said third reactive unit and said second reactive unit is incompatible with the reaction between said first reactive unit and said second reactive unit.

23. An in vitro method of increasing reaction selectivity among a plurality of reactants in a nucleic acid-templated synthesis to produce, without the assistance of the ribosome, a reaction product that is not a nucleic acid, the method comprising the steps of:

(a) providing (i) a template associated with a capturable moiety and comprising a first reactive unit covalently affached to a first oligonucleotide comprising a predetermined codon sequence, (ii) a first transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon sequence capable of annealing to said codon sequence, and (iii) a second transfer unit comprising a third reactive unit different from said second reactive unit associated with a third oligonucleotide without an anti-codon sequence capable of annealing to said codon sequence; and

(b) mixing said template, said first transfer unit and said second transfer unit under conditions to permit annealing of said second oligonucleotide of said first transfer unit to said first oligonucleotide of said template thereby to enhance covalent bond formation between said second reactive unit and said first reactive unit to produce the reaction product relative to covalent bond formation between said third reactive unit and said first reactive unit,

wherein the reaction between said second reactive unit and said third reactive unit are incompatible with at least one of their reactions with said first reactive unit.

24. The method of claim 23, wherein said first transfer unit is associated with a capturable moiety.

25. The method of claim 23, wherein said second transfer unit is associated with a capturable moiety.

26. The method of claim 23, 24, 25, wherein said capturable moiety is selected from the group consisting of biotin, avidin and streptavidin.

27. The method of claim 26, further comprising the step of capturing said capturable moiety.

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

29. The method of claim 23, wherein said third reactive unit is covalently attached to said third oligonucleotide.

30. The method of claim 23, wherein said second reactive unit and said third reactive unit are capable of reacting independently with said first reactive unit.

31. The method of claim 23, comprising providing a plurality of transfer units.

32. The method of claim 23, further comprising:

providing a second template comprising a fourth reactive unit associated with a fourth oligonucleotide comprising a second predetermined codon sequence, different from said predetermined codon sequence of said first oligonucleotide, wherein said second predetermined codon sequence is capable of annealing with said third oligonucleotide; and

mixing said second template with said first transfer unit, said second transfer unit, and said template comprising said first reactive unit associated with said first oligonucleotide under conditions to permit

annealing of said second oiigonucieotide of said first transfer unit to said first oligonucleotide of said template and, in the same solution,

annealing of said third oligonucleotide of said second transfer unit to said fourth oligonucleotide of said second template, thereby to induce covalent bond formation both between said second reactive unit and said first reactive unit and between said fourth reactive unit and said third reactive unit.