Evolving new molecular function

• US 7,771,935 B2
• Filed: 09/24/2004
• Issued: 08/10/2010
• 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: Calderone, Christopher T., Liu, David R., Gartner, Zev J.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Thomas; David C
• Application Number:
  US10/950,367
• Publication Number:
  US 20050170376A1
• Time in Patent Office: 2,146 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 inducing a reaction between first and second reactive units during a nucleic acid-templated chemical reaction, the method comprising the steps of:

(a) providing (i) a template comprising a first reactive unit covalently attached to a first oligonucleotide comprising a codon and (ii) a transfer unit comprising a second reactive unit covalently associated with a second oligonucleotide comprising an anti-codon capable of annealing to said codon, wherein said codon or said anti-codon comprise first and second spaced apart regions; and

(b) annealing said first and second oligonucleotides thereby to bring said first reactive unit and said second reaction unit into reactive proximity and produce a reaction product that is not a nucleic acid, wherein, upon annealing, said codon or said anti-codon having said first and second spaced apart regions anneal to the corresponding anti-codon or codon via said first and second spaced apart regions to produce a loop of oligonucleotides disposed between said first and second spaced apart regions that are not annealed to the corresponding anti-codon or codon, wherein the loop of oligonucleotides permits the codon to anneal to the corresponding anti-codon and bring the first and second reactive units into reactive proximity.

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

3. The method of claim 2, wherein each of said reactive units is attached adjacent a terminal portion of its corresponding oligonucleotide.

4. The method of claim 1, 2, or 3, wherein said codon or said anti-codon is disposed at least 10 bases away from its corresponding reactive unit.

5. The method of claim 1, 2, or 3, wherein said codon or said anti-codon is disposed at least 20 bases away from its corresponding reactive unit.

6. The method of claim 1, 2, or 3, wherein said codon or said anti-codon is disposed directly adjacent its corresponding reactive unit.

7. The method of claim 1, wherein in said codon or said anti-codon comprising said first and second spaced apart regions, said first region is disposed directly adjacent a terminus of its corresponding oligonucleotide.

8. The method of claim 1 or 7, wherein said first region of said codon or said anti-codon comprises three, four or five adjacent nucleotides.

9. The method of claim 1 or 7, wherein said first region of said codon or said anti-codon comprises five adjacent nucleotides.

10. The method of claim 1 or 7, wherein said second region is disposed at least 20 bases away from said reactive unit.

11. The method of claim 1 or 7, wherein said second region is disposed at least 30 bases away from said reactive unit.

12. An in vitro method of inducing a reaction between first and second reactive units during a nucleic acid-templated chemical reaction, the method comprising the steps of:

(a) providing (i) a template comprising a first reactive unit covalently attached to a first oligonucleotide having a proximal end and a distal end and comprising a codon and (ii) a transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon capable of annealing with said codon, wherein said first reactive unit is attached to an attachment site intermediate said proximal end and said distal end of said first oligonucleotide; and

(b) annealing said oligonucleotides together thereby to bring said first reactive unit and said second reactive unit into reactive proximity and produce a reaction product that is not a nucleic acid.

13. The method of claim 12, wherein said template comprises a second, different codon capable of annealing to a second, different anti-codon sequence.

14. The method of claim 13, wherein said first codon is located proximal to, and said second codon is located distal to, said attachment site of said first reactive unit.

15. The method of claim 13 or 14, further comprising providing a second transfer unit comprising a third reactive unit associated with a third oligonucleotide comprising a second, different anti-codon sequence capable of annealing with said second codon.

16. The method of claim 15, wherein said first anti-codon of said first transfer unit anneals to said first codon of said template and said second anti-codon of said second transfer unit anneals to said second codon of said template.

17. The method of claim 16, wherein said first transfer unit anneals with said template concurrently with said second transfer unit, so that said second reactive unit and said third reactive unit react with said first reactive unit.

18. The method of claim 12, wherein said second reactive unit is covalently attached to said second oligonucleotide.

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

20. The method of claim 12, wherein said first reactive unit is a scaffold molecule.