REACTION DISCOVERY SYSTEM

• US 20110312507A1
• Filed: 01/09/2008
• Published: 12/22/2011
• Est. Priority Date: 07/15/2005
• Status: Active Grant

• Current Assignee: United States Navy (U.S. Department Of Defense)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Liu, David R., Rozenman, Mary M., Kanan, Matthew William
• Granted Patent: US 8,691,729 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• 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

Abstract

A novel reaction discovery system that does not depend on DNA duplex formation is provided. The advantages of this system include exploring reactions conditions not possible where DNA hybridization is required. For example, the inventive reaction discovery system allows for reaction conditions using organic solvents, higher temperatures, and water-insoluble reagents, catalysts, and ligands. The invention also provides single-stranded oligonucleotide templates with substrate pairs covalently attached and methods of screening for reaction conditions that result in a direct covalent bond between the substrates. Kits are also provided for practicing this novel reaction discovery system.

Claims

1. A method of identifying new chemical reactions, the method comprising steps of:

providing one or more templates, which one or more templates have a first and second substrate associated therewith and the one or more templates including sequences that identify the substrates associated therewith;

subjecting the templates with associated substrates to reaction conditions; and

identifying a reaction product between the two substrates associated with the template.

2. The method of claim 1, wherein the template comprises a nucleic acid.

3. The method of claim 1, wherein the template comprises DNA or RNA.

4. The method of claim 1, wherein the template comprises DNA.

5. The method of claim 1, wherein one of the two substrates is attached to the end of the template.

6. The method of claim 1, wherein one of the two substrates is attached to the template through an internal nucleotide.

7. The method of claim 1, wherein the two substrates are within 20 bases of each other.

8. The method of claim 1, wherein the two substrates are within 15 bases of each other.

9. The method of claim 1, wherein the two substrates are within 10 bases of each other.

10. The method of claim 1, wherein the substrates are covalently associated with the template.

11. The method of claim 1, wherein the methods does not require the hybridization of oligonucleotides.

12. The method of claim 1, wherein the step of subjecting the template comprises heating, cooling, adding a catalyst, adding a reagent, changing pH, increasing pressure, decreasing pressure, and adding a solvent.

13. The method of claim 1, wherein the step of subjecting the template comprises adding a stereoselective catalyst.

14. The method of claim 1, wherein the template further comprises an affinity reagent.

15. The method of claim 14, wherein the affinity reagent is biotin.

16. The method of claim 14, wherein the first substrate and the affinity reagent are attached to the template through a cleavable linker.

17. The method of claim 16, wherein the cleavable linker is a disulfide bond.

18. The method of claim 15, wherein the step of identifying comprises selecting templates covalently bound to biotin through the covalently attached substrates using streptavidin.

19. The method of claim 1, wherein the step of identifying comprises using DNA microarray to identify substrate pairs that formed a reaction product under the reaction conditions.

20. A method of identifying new chemical reactions, the method comprising steps of:

providing one or more DNA templates,

wherein one or more templates have two substrates associated therewith;

wherein a portion of the sequence of the DNA template identifies the substrates associated with the template; and

wherein one of the two substrates and biotin is linked to the template through a cleavable linker;

subjecting the templates with substrates attached thereto to reaction conditions,

whereby a covalent bond is formed between the substrates associated with the template;

cleaving the linker;

selecting substrate pairs that reacted to form a covalent bond between the substrates using streptavidin; and

identifying the selected substrate pairs and reaction conditions.

21. The method of claim 20, wherein the step of identifying comprises:

amplifying the DNA template associated the identified reaction product using PCR; and

sequencing the amplified DNA.

22. The method of claim 20, wherein the step of identifying comprises using a microarray to identify the selected substrate pairs that reacted.

23. A template oligonucleotide for reaction discovery comprising:

an oligonucleotide with sequences identifying substrates attached thereto;

two substrates; and

a cleavable linker with an affinity reagent.

24. The template of claim 23, wherein the oligonucleotide is DNA.

25. The template of claim 23, wherein the oligonucleotide further comprises primer sequences.

26. The template of claim 23, wherein the cleavable linker is a disulfide bond.

27. The template of claim 23, wherein the affinity reagent is biotin.

30. A kit for identifying new chemical reactions comprising one or more template oligonucleotides of claim 23 and a DNA microarray.

31. The kit of claim 30 further comprising any of the reagents selected from the group consisting of enzymes, solvents, reagents, buffers, and streptavidin beads.

28. A method for preparing a template oligonucleotide for reaction discovery, the method comprising steps of:

providing a template oligonucleotide with a first substrate attached and with a sequence that identifies the first attached substrate;

hybridizing through an overhand an oligonucleotide with a sequence that identifies a second substrate;

polymerizing off the template oligonucleotide by primer extension to include the sequence that identifies the second substrate;

denaturing resulting duplex; and

ligating onto the template oligonucleotide an oligonucleotide with the second substrate and affinity reagent linked to the oligonucleotide through a cleavable linker.

29. The method of claim 28, further comprising purifying the template oligonucleotide.