Methods for identifying compounds of interest using encoded libraries

• US 20070224607A1
• Filed: 10/23/2006
• Published: 09/27/2007
• Est. Priority Date: 10/28/2005
• Status: Active Grant

• Current Assignee: GlaxoSmithKline LLC (GlaxoSmithKline PLC)
• Original Assignee: Praecis Pharmaceuticals, Inc. (GlaxoSmithKline PLC)
• Inventors: Clark, Matthew, Wagner, Richard, Hale, Stephen, Morgan, Barry, Benjamin, Dennis, Israel, David, Hansen, Nils, Kavarana, Malcolm, Centrella, Paolo, Arico-Muendel, Christopher, Acharya, Raksha, Gefter, Malcolm, Creaser, Steffen, Franklin, George
• Granted Patent: US 7,989,395 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• 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

The present invention provides a method for identifying a compound of interest by screening libraries of molecules which include an encoding oligonucleotide tag.

Claims

1. A method for identifying one or more compounds which bind to a biological target, said method comprising:

(A) synthesizing a library of compounds, wherein the compounds comprise a functional moiety comprising two or more building blocks which is operatively linked to an initial oligonucleotide which identifies the structure of the functional moiety by;

(i) providing a solution comprising m initiator compounds, wherein m is an integer of 1 or greater, where the initiator compounds consist of a functional moiety comprising n building blocks, where n is an integer of 1 or greater, which is operatively linked to an initial oligonucleotide which identifies the n building blocks;

(ii) dividing the solution of step (i) into r reaction vessels, wherein r is an integer of 2 or greater, thereby producing r aliquots of the solution;

(iii) reacting the initiator compounds in each reaction vessel with one of r building blocks, thereby producing r aliquots comprising compounds consisting of a functional moiety comprising n+1 building blocks operatively linked to the initial oligonucleotide; and

(iv) reacting the initial oligonucleotide in each aliquot with one of a set of r distinct incoming oligonucleotides in the presence of an enzyme which catalyzes the ligation of the incoming oligonucleotide and the initial oligonucleotide, under conditions suitable for enzymatic ligation of the incoming oligonucleotide and the initial oligonucleotide;

thereby producing r aliquots of molecules consisting of a functional moiety comprising n+1 building blocks operatively linked to an elongated oligonucleotide which encodes the n+1 building blocks;

(B) contacting the biological target with the library of compounds, or a portion thereof, under conditions suitable for at least one member of the library of compounds to bind to the target;

(C) removing library members that do not bind to the target;

(D) sequencing the encoding oligonucleotides of the at least one member of the library of compounds which binds to the target, and

(E) using the sequences determined in step (D) to determine the structure of the functional moieties of the members of the library of compounds which bind to the biological target, thereby identifying one or more compounds which bind to the biological target.

2. The method of claim 1, further comprising amplifying the encoding oligonucleotides of the at least one member of the library of compounds which binds to the target.

3. The method of claim 2, wherein said amplifying step comprises:

(i) forming a water-in-oil emulsion to create a plurality of aqueous microreactors, wherein at least one of the microreactors comprises the at least one member of the library of compounds that binds to the target, a single bead capable of binding to the encoding oligonucleotide of the at least one member of the library of compounds that binds to the target, and amplification reaction solution containing reagents necessary to perform nucleic acid amplification;

(ii) amplifying the encoding oligonucleotide in the microreactors to form amplified copies of said encoding oligonucleotide; and

(iii) binding the amplified copies of the encoding oligonucleotide to the beads in the microreactors.

4. The method of claim 1, wherein said sequencing step (D) comprises:

(i) annealing an effective amount of a sequencing primer to the amplified copies of the encoding oligonucleotide and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto a 3′

end of said sequencing primer, a sequencing reaction byproduct; and

(ii) identifying the sequencing reaction byproduct, thereby determining the sequence of the encoding oligonucleotide.

8. The method of any one of claims 1, 4, or 5, wherein said sequencing of the encoding oligonucleotides is carried out by a pyrophosphate-based sequencing reaction or a single molecule sequencing by synthesis method.

9. The method of claim 8, wherein the sequencing reaction byproduct is PPi and a coupled sulfurylase/luciferase reaction is used to generate light for detection.

10. The method of any one of claims 1 or 5, further comprising the step of enriching for beads which bind amplified copies of the encoding oligonucleotide away from beads to which no encoding oligonucleotide is bound.

11. The method of claim 10, wherein the method for said enrichment step is selected from the group consisting of affinity purification, and electrophoresis.

12. The method of claim 3, further comprising breaking the emulsion to retrieve one or more of the amplified copies of the encoding oligonucleotide.

13. The method of claim 1 or 5, further comprising the step of

(A)(v) combining two or more of the r aliquots, thereby producing a solution comprising molecules consisting of a functional moiety comprising n+1 building blocks, which is operatively linked to an elongated oligonucleotide which encodes the n+1 building blocks.

14. The method of claim 13, wherein r aliquots are combined.

15. The method of claim 13, wherein the steps (A)(i) to (A)(v) are conducted one or more times to yield cycles 1 to i, where i is an integer of 2 or greater, wherein in cycle s+1, where s is an integer of i-1 or less, the solution comprising m initiator compounds of step (a) is the solution of step (e) of cycle s.

16. The method of of claim 1 or 5, wherein at least one of building blocks is an amino acid.

17. The method of claim 1 or 5, wherein the initial oligonucleotide is a covalently coupled double-stranded oligonucleotide.

18. The method of claim 17, wherein the incoming oligonucleotide is a double-stranded oligonucleotide.

19. The method of claim 1 or 5, wherein the initiator compounds comprise a linker moiety comprising a first functional group adapted to bond with a building block, a second functional group adapted to bond to the 5′

end of an oligonucleotide, and a third functional group adapted to bond to the 3′

-end of an oligonucleotide.

20. The method of claim 19, wherein the linker moiety is of the structure

21. The method of claim 20, wherein:

A is an amino group;

B is a phosphate group; and

C is a phosphate group.

22. The method of claim 20, wherein D, E and F are each, independently, an alkylene group or an oligo(ethylene glycol) group.

23. The method of claim 20, wherein S is a carbon atom, a nitrogen atom, a phosphorus atom, a boron atom, a phosphate group, a cyclic group or a polycyclic group.

24. The method of claim 23, wherein the linker moiety is of the structure

25. The method of claim 24, wherein each of n, m and p is independently an integer from 2 to eight.

26. The method of claim 25, wherein each of n, m and p is independently an integer from 3 to 6.

27. The method of claim 24, wherein the linker moiety has the structure

28. The method of claim 1 or 5, wherein each of said initiator compounds comprises a reactive group and wherein each of said r building blocks comprises a complementary reactive group which is complementary to said reactive group.

29. The method of claim 28, wherein the reactive group and the complementary reactive group are selected from the group consisting of an amino group;

a carboxyl group;

a sulfonyl group;

a phosphonyl group;

an epoxide group;

an aziridine group; and

an isocyanate group.

30. The method of claim 28, wherein reactive group and the the complementary reactive group are selected from the group consisting of a hydroxyl group;

a carboxyl group;

a sulfonyl group;

a phosphonyl group;

an epoxide group;

an aziridine group; and

an isocyanate group.

31. The method of claim 28, wherein the reactive group and the complementary reactive group are selected from the group consisting of an amino group and an aldehyde or ketone group.

32. The method of claim 28, wherein the reaction between the reactive group and the complementary reactive group is conducted under reducing conditions.

33. The method of claim 28, wherein the reactive group and the complementary reactive group are selected from the group consisting of a phosphorous ylide group and an aldehyde or ketone group.

34. The method of claim 28, wherein the reactive group and the complementary reactive group react via cycloaddition to form a cyclic structure.

35. The method of claim 34, wherein the reactive group and the complementary reactive group are selected from the group consisting of an alkyne and an azide.

36. The method of claim 28, wherein the reactive group and the complementary functional group are selected from the group consisting of a halogenated heteroaromatic group and a nucleophile.

37. The method of claim 36, wherein the halogenated heteroaromatic group is selected from the group consisting of chlorinated pyrimidines, chlorinated triazines and chlorinated purines.

38. The method of claim 36, wherein the nucleophile is an amino group.

39. The method of claim 13, further comprising following cycle i, the step of:

(A)(vi) cyclizing one or more of the functional moieties.

40. The method of claim 39, wherein a functional moiety of step (A)(vi) comprises an azido group and an alkynyl group.

41. The method of claim 40, wherein the functional moiety is maintained under conditions suitable for cycloaddition of the azido group and the alkynyl group to form a triazole group, thereby forming a cyclic functional moiety

42. The method of claim 41, wherein the cycloaddition reaction is conducted in the presence of a copper catalyst.

43. The method of claim 42, wherein at least one of the one or more functional moieties of step (f) comprises at least two sulfhydryl groups, and said functional moiety is maintained under conditions suitable for reaction of the two sulfhydryl groups to form a disulfide group, thereby cyclicizing the functional moiety.

44. The method of claim 1 or 5, wherein the initial oligonucleotide comprises a PCR primer sequence.

45. The method of claim 13, wherein the incoming oligonucleotide of cycle i comprises a PCR closing primer.

46. The method of claim 13, further comprising following cycle i, the step of (

d) ligating an oligonucleotide comprising a closing PCR primer sequence to the encoding oligonucleotide.

47. The method of claim 46, wherein the oligonucleotide comprising a closing PCR primer sequence is ligated to the encoding oligonucleotide in the presence of an enzyme which catalyzes said ligation.

5. A method for identifying one or more compounds which bind to a biological target, said method comprising:

(A) synthesizing a library of compounds, wherein the compounds comprise a functional moiety comprising two or more building blocks which is operatively linked to an initial oligonucleotide which identifies the structure of the functional moiety by;

(i) providing a solution comprising m initiator compounds, wherein m is an integer of 1 or greater, where the initiator compounds consist of a functional moiety comprising n building blocks, where n is an integer of 1 or greater, which is operatively linked to an initial oligonucleotide which identifies the n building blocks;

(ii) dividing the solution of step (i) into r reaction vessels, wherein r is an integer of 2 or greater, thereby producing r aliquots of the solution;

(iii) reacting the initiator compounds in each reaction vessel with one of r building blocks, thereby producing r aliquots comprising compounds consisting of a functional moiety comprising n+1 building blocks operatively linked to the initial oligonucleotide; and

(iv) reacting the initial oligonucleotide in each aliquot with one of a set of r distinct incoming oligonucleotides in the presence of an enzyme which catalyzes the ligation of the incoming oligonucleotide and the initial oligonucleotide, under conditions suitable for enzymatic ligation of the incoming oligonucleotide and the initial oligonucleotide;

thereby producing r aliquots of molecules consisting of a functional moiety comprising n+1 building blocks operatively linked to an elongated oligonucleotide which encodes the n+1 building blocks;

(B) contacting the biological target with the library of compounds, or a portion thereof, under conditions suitable for at least one member of the library of compounds to bind to the target;

(C) removing library members that do not bind to the target;

(D) sequencing the encoding oligonucleotides of the at least one member of the library of compounds which binds to the target, wherein said sequencing comprises;

(i) annealing an effective amount of a sequencing primer to the amplified copies of the encoding oligonucleotide and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto a 3′

end of said sequencing primer, a sequencing reaction byproduct; and

(ii) identifying the sequencing reaction byproduct, thereby determining the sequence of the encoding oligonucleotide; and

(E) using the sequence of the encoding oligonucleotide determined in step (D) to determine the structure of the functional moieties of the members of the library of compounds which bind to the biological target, thereby identifying one or more compounds which bind to the biological target.

6. The method of claim 5, further comprising amplifying the encoding oligonucleotides of the at least one member of the library of compounds which binds to the target.

7. The method of claim 6, wherein said amplification of the encoding oligonucleotides is carried out by a method selected from the group consisting of:

the polymerase chain reaction (PCR);

transcription-based amplification, rapid amplification of cDNA ends, continuous flow amplification, and rolling circle amplification.