Evolving new molecular function

• US 8,206,914 B2
• Filed: 07/12/2010
• Issued: 06/26/2012
• Est. Priority Date: 08/19/2002
• 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., Gartner, Zev J., Doyon, Jeffrey B., Calderone, Christopher T., Kanan, Matthew W., Li, Xiaoyu, Snyder, Thomas M., Rosenbaum, Daniel M.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US12/834,072
• Publication Number:
  US 20110190141A1
• Time in Patent Office: 715 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 enriching a product of a nucleic acid-templated synthesis, the method comprising the steps of:

(a) providing a first library of molecules comprising a plurality of reaction products of a nucleic acid templated synthesis, which are not nucleic acids, wherein each reaction product is covalently attached to a corresponding oligonucleotide that templated the synthesis of the reaction product, and wherein each oligonucleotide comprises a nucleotide sequence indicative of the reaction product associated therewith, and wherein a portion of said reaction products are capable of binding to a preselected binding moiety;

(b) exposing said first library of molecules to said binding moiety under conditions to permit reaction product capable of binding said binding moiety to bind thereto, wherein the reaction product has a K_d for the binding moiety of no less than 0.9 nM;

(c) removing unbound reaction products; and

(d) eluting bound reaction product from said binding moiety to produce a second library of molecules enriched at least 50-fold for reaction product that binds said binding moiety relative to said first library.

2. The method of claim 1, wherein in step (b), said binding moiety is immobilized on a solid support.

3. The method of claim 1, wherein said binding moiety is a target biomolecule.

4. The method of claim 3, wherein said target biomolecule is a protein.

5. The method of claim 1, wherein in step (d), said second library is enriched at least 100-fold for reaction product that binds said binding moiety.

6. The method of claim 5, wherein in step (d), said second library is enriched at least 1,000-fold for reaction product that binds said binding moiety.

7. The method of claim 1, further comprising repeating steps (b), (c), and (d).

8. The method of claim 7, wherein repeating steps (b), (c), and (d) produces a third library enriched by at least 10,000-fold for reaction product that binds said binding moiety.

9. The method of claim 8, wherein said library is enriched by at least 100,000-fold for reaction product that binds said binding moiety.

10. The method of claim 1, wherein said oligonucleotide comprises a first sequence that identifies a first reactive unit that produced said reaction product capable of binding said preselected binding moiety.

11. The method of claim 10, wherein said oligonucleotide comprises a second sequence that identifies a second reactive unit that produced said reaction product capable of binding said preselected binding moiety.

12. The method of claim 1, comprising the additional step of amplifying oligonucleotide associated with the enriched reaction product.

13. The method of claim 1, comprising the additional step of determining the sequence of the oligonucleotide associated with the enriched reaction product.

14. The method of claim 12, comprising the additional step of determining the sequence of the amplified oligonucleotide.

15. The method of claim 13, further comprising the step of characterizing said reaction product from information in said sequence of said oligonucleotide.

16. The method of claim 15, further comprising the step of identifying a new chemical reaction that produced said reaction product.

17. The method of claim 14, further comprising the step of characterizing the reaction product from information in said sequence of said oligonucleotide.

18. The method of claim 17, further comprising the step of identifying a new chemical reaction that produced said reaction product.

19. An in vitro method of enriching a product of a nucleic acid-templated synthesis, the method comprising the steps of:

(a) providing a first library of molecules comprising a plurality of reaction products of a nucleic acid templated synthesis, which are not nucleic acids, wherein each reaction product is covalently attached to a corresponding oligonucleotide that templated the synthesis of the reaction product, wherein each oligonucleotide comprises a nucleotide sequence indicative of the reaction product associated therewith, wherein no oligonucleotide is linked by a direct or indirect covalent or non-covalent interaction to a capturable moiety selected from the group consisting of biotin, avidin and streptavidin; and

wherein a portion of said reaction products are capable of binding to a preselected binding moiety;

(b) exposing said first library of molecules to said binding moiety under conditions to permit reaction product capable of binding said binding moiety to bind thereto;

(c) removing unbound reaction products; and

(d) eluting bound reaction product from said binding moiety to produce a second library of molecules enriched at least 50-fold for reaction product that binds said binding moiety relative to said first library.