Methods for synthesis of encoded libraries

• US 8,410,028 B2
• Filed: 03/18/2011
• Issued: 04/02/2013
• Est. Priority Date: 12/17/2003
• Status: Active Grant

• Current Assignee: GlaxoSmithKline LLC (GlaxoSmithKline PLC)
• Original Assignee: GlaxoSmithKline LLC (GlaxoSmithKline PLC)
• Inventors: Morgan, Barry, Hale, Stephen, Arico-Muendel, Christopher C., Clark, Matthew, Wagner, Richard, Israel, David I., Gefter, Malcolm L., Benjamin, Dennis, Hansen, Nils Jakob Vest, Kavarana, Malcolm J., Franklin, George J., Centrella, Paolo A., Acharya, Raksha A., Creaser, Steffan Phillip
• Primary Examiner(s): Joike, Michele K
• Assistant Examiner(s): GALISTEO GONZALEZ, ANTONIO
• Application Number:
  US13/051,314
• Publication Number:
  US 20110251089A1
• Time in Patent Office: 746 Days
• Field of Search: 506/26, 506/28, 506/27
• US Class Current: 506/27
• 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
• CPC Class Codes: C40B 50/10 involving encoding steps

Abstract

The present invention provides a method of synthesizing 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 the steps of:

(a) contacting the biological target with a library of compounds under conditions suitable for at least one member of the compound library to bind to the target, wherein each compound of the library comprises a functional moiety comprising one or more building blocks, said moiety operatively linked to an encoding oligonucleotide which identifies the structure of the functional moiety;

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

(c) amplifying the encoding oligonucleotide of the at least one member of the compound library which binds to the target;

(d) sequencing the encoding oligonucleotide of step (c); and

(e) using the sequence determined in step (d) to determine the structure of the functional moiety of the at least one member of the compound library which binds to the biological target;

said library of compounds synthesized by a method comprising the steps of;

(i) providing a solution comprising m initiator compounds, wherein m is an integer of 1 or greater, wherein the initiator compounds comprise a reactive group and consist of an initial 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;

wherein the initial functional moiety and the initial oligonucleotide are linked by a linking moiety and wherein the initial oligonucleotide is double-stranded and the linker moiety is covalently coupled to the initial functional moiety and to both strands of the initial oligonucleotide;

(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, said building blocks comprising at least one complementary reactive group, wherein the at least one complementary is complementary to the reactive group of step (i), under conditions suitable to form a covalent bond, 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 corresponding to the building block of step (iii) 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 to form an encoding oligonucleotide, wherein the last of the incoming oligonucleotides comprises a capping sequence, said capping sequence comprising a nucleotide sequence containing degenerate nucleotides;

thereby producing r aliquots comprising molecules consisting of a functional moiety comprising n+1 building blocks operatively linked to an encoding oligonucleotide which identifies the structure of the functional moiety comprising the n+1 building blocks,

thereby identifying one or more compounds which bind to the biological target.

2. The method of claim 1, further comprising the step of

(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.

3. The method of claim 2 wherein r aliquots are combined.

4. The method of claim 2 wherein the steps (i) to (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.

5. The method of either claim 2 or 1, wherein in at least one of cycles 1 to i step (d) precedes step (c).

6. The method of claim 2 or 1 wherein at least one of the building blocks is an amino acid.

7. The method of claim 1, wherein the enzyme is DNA ligase, RNA ligase, DNA polymerase, RNA polymerase or topoisomerase.

8. The method of claim 1, wherein the incoming oligonucleotide is a double-stranded oligonucleotide.

9. The method of claim 2 or 1 wherein the linking moiety comprises a first functional group adapted to bond with one of the n building blocks, a second functional group adapted to bond to the 5′

end of one strand of the initial oligonucleotide, and a third functional group adapted to bond to the 3′

-end of the other strand of the initial oligonucleotide wherein the linking moiety is of the structure

10. The method of claim 9 wherein:

A is an amino group;

B is a phosphate group; and

C is a phosphate group.

11. The method of claim 9 wherein D, E and F are each, independently, an alkylene group or an oligo(ethylene glycol) group.

12. The method of claim 9 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.

13. The method of claim 12 wherein the linking moiety is of the structure

14. The method of claim 13 wherein each of n, m and p is independently an integer from 2 to eight.

15. The method of claim 14 wherein each of n, m and p is independently an integer from 3 to 6.

16. The method of claim 13 wherein the linking moiety has the structure

17. The method of claim 1, wherein the reactive group is an amino group and the complementary reactive group is selected from the group consisting of a carboxyl group;

a sulfonyl group;

a phosphonyl group;

an epoxide group;

an aziridine group; and

an isocyanate group.

18. The method of claim 1, wherein reactive group or the complementary reactive group is independently 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.

19. The method of claim 1, wherein the reactive group is an amino group and the complementary reactive group is selected from the group consisting of an aldehyde group and a ketone group.

20. The method of claim 1, wherein the reaction between the reactive group and the complementary reactive group is conducted under reducing conditions.

21. The method of claim 1, wherein the reactive group or the complementary reactive group is independently selected from the group consisting of a phosphorous ylide group, an aldehyde group, and a ketone group.

22. The method of claim 1, wherein the reactive group and the complementary reactive group react via cycloaddition to form a cyclic structure.

23. The method of claim 22, wherein the reactive group or the complementary reactive group is independently selected from the group consisting of an alkyne and an azide.

24. The method of claim 1, wherein the reactive group or the complementary functional group is independently selected from the group consisting of a halogenated heteroaromatic group and a nucleophile.

25. The method of claim 24 wherein the halogenated heteroaromatic group is selected from the group consisting of chlorinated pyrimidines, chlorinated triazines and chlorinated purines.

26. The method of claim 24 wherein the nucleophile is an amino group.

27. The method of claim 2, further comprising following cycle i, the step of:

(f) cyclizing one or more of the functional moieties.

28. The method of claim 24 wherein a functional moiety of step (f) comprises an azido group and an alkynyl group.

29. The method of claim 28 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.

30. The method of claim 28 wherein the cycloaddition reaction is conducted in the presence of a copper catalyst.

31. The method of claim 30 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.

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

33. The method of claim 4, wherein the incoming oligonucleotide of cycle i comprises a PCR closing primer.

34. The method of claim 4, further comprising following cycle i, the step of

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

35. The method of claim 34 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.

36. The method of claim 1, wherein the reactions for synthesizing the compound library are conducted in solution.

37. The method of claim 1, wherein the reactions for synthesizing the compound library are conducted in a mixed aqueous/organic solution.

38. The method of claim 1, wherein the synthesized molecule is a polymeric compound.

39. The method of claim 1, wherein the synthesized molecule is a non-polymeric compound.

63. The method of claim 1 or 40, wherein said capping sequence comprises SEQ ID NO:

894;

3′

-AA GTCGCAAGCT NNNNN GTCTGTTCGAAGTGGACG-5′

, wherein N is any one of A, T, G, or C.

64. The method of claim 1 or 40, wherein said capping sequence comprises SEQ ID NO.:

897;

3′

-AA GTCGCAAGCTACG ABBBABBBABBBA GACTACCGCGCTCCCTCCG-5′

, wherein B is any one of T, G, or C.

40. A method for identifying a compound which binds to a biological target, said method comprising the steps of

(a) contacting the biological target with a compound library comprising at least about 10² distinct compounds, said compounds comprising a functional moiety comprising one or more building blocks which is operatively linked by a linking moiety to an encoding oligonucleotide which identifies the structure of the functional moiety under conditions suitable for at least one member of the compound library to bind to the target;

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

(c) amplifying the encoding oligonucleotide of the at least one member of the compound library which binds to the target;

(d) sequencing the encoding oligonucleotide of step (c); and

(e) using the sequence determined in step (d) to determine the structure of the functional moiety operatively linked to by a linking moiety to the encoding oligonucleotide of the at least one member of the compound library which binds to the biological target;

wherein said library comprises a multiplicity of compounds which are each of Formula I;

41. The method of claim 40 wherein the library comprises at least about 10⁵ copies of each of the distinct compounds.

42. The method of claim 40 wherein the library comprises at least about 10⁶ copies of each of the distinct compounds.

43. The method of claim 40 wherein the library comprises at least about 10⁴ distinct compounds.

44. The method of claim 40 wherein the library comprises at least about 10⁶ distinct compounds.

45. The method of claim 40 wherein the library comprises at least about 10⁸ distinct compounds.

46. The method of claim 40 wherein the library comprises at least about 10¹⁰ distinct compounds.

47. The method of claim 40 wherein the compound library comprises at least about 10¹² distinct compounds.

48. The method of claim 40, wherein A, B, C, D, E, F and S each have the same identity for each compound of Formula I.

49. The method of claim 40, wherein the compound library consists essentially of a multiplicity of compounds of Formula I.

50. The method of claim 40, wherein D, E and F are each independently an alkylene chain or an oligo(ethylene glycol) chain.

51. The method of claim 40, wherein Y and Z are substantially complementary and are oriented in the compound so as to enable Watson-Crick base pairing and duplex formation under suitable conditions.

52. The method of claim 40, wherein Y and Z are the same length or different lengths.

53. The method of claim 52 wherein Y and Z are the same length.

54. The method of claim 40, wherein Y and Z are each 10 or more bases in length and have complementary regions of ten or more base pairs.

55. The method of claim 40, wherein S is a carbon atom, a boron atom, a nitrogen atom, a phosphorus atom, or a polyatomic scaffold.

56. The method of claim 40, wherein S is a phosphate group or a cyclic group.

57. The method of claim 56 wherein S is a cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl group.

58. The method of claim 40, wherein the linking moiety is of the structure

59. The method of claim 58 wherein each of n, m and p is independently an integer from 2 to eight.

60. The method of claim 59 wherein each of n, m and p is independently an integer from 3 to 6.

61. The method of claim 60 wherein the linking moiety has the structure

62. The method of claim 40, wherein X and Z comprise a PCR primer sequence.