Method for selecting a chemical entity from a tagged library

• US 20060292603A1
• Filed: 04/13/2006
• Published: 12/28/2006
• Est. Priority Date: 10/30/2002
• Status: Active Grant

• Current Assignee: Nuevolution A/S (Amgen Inc.)
• Original Assignee: Nuevolution A/S (Amgen Inc.)
• Inventors: Franch, Thomas, Gouliaev, Alex Haahr
• Granted Patent: US 8,722,583 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C40B 50/10 involving encoding steps
• CPC Class Codes: C40B 50/16 involving encoding steps

Abstract

Disclosed is a method for determining the identity of a chemical entity having a preselected property. The chemical entity is identified from a library composed of a plurality of different chemical entities each appended to unique identifier tags. An anti-tag having the capability of specifically interacting with the unique identifier tag is recovered during the method and used for identification purposes.

Claims

1. In a method of determining the identity of a chemical entity in a combinatorial library comprising a plurality of structurally distinct chemical entities,

wherein each chemical entity is tagged with an entity tag identifying the structure of the chemical entity,

wherein each chemical entity comprises a plurality of fragments,

wherein the entity tag consists essentially of a plurality of fragment tags, each fragment tag identifying one of the fragments constituting a fragment of the chemical entity tagged by said entity tag,

the improvement comprising

ligating at least one pair of fragment tags by enzyme-catalysed ligation.

2. The method of claim 1 wherein the tagged entity is obtained by initially providing a tagged first fragment, said tagged first fragment comprising a first fragment and a first fragment tag which identifies said fragment, and, sequentially or simultaneously, combining said first fragment with one or more further fragments and combining said first fragment tag with one or more further fragment tags, wherein each further fragment tag identifies a further fragment.

3. The method of claim 2 which comprises

(1) directly attaching a first fragment tag to a first fragment,

(2) ligating a second fragment tag to the first fragment tag, and

(3) ligating a second fragment to the first fragment,

wherein steps (2) and (3) may be simultaneous or may be interchanged, and

wherein as a result of steps (2) and (3), the second fragment tag is indirectly attached, through said first fragment tag and first fragment, to said second fragment.

4. The method of claim 3 which further comprises ligating a third fragment to the second fragment, and a third fragment tag to the second fragment tag, simultaneously or sequentially.

5. The method of claim 2 wherein the fragment tags are combined solely by enzyme-catalyzed ligation.

6. The method of claim 2 wherein the fragments are combined solely by chemical (non-enzyme-catalyzed) reaction.

7. The method of claim 1 wherein the library entities are oligonucleotides.

8. The method of claim 7 wherein the oligonucleotides are selected from the group consisting of DNA, RNA, LNA, and PNA oligomers.

9. The method of claim 1 wherein the library entities are peptides.

10. The method of claim 9 wherein the fragments identified by the fragment tags are individual amino acid residues.

11. The method of claim 10 wherein the individual amino acid residues are naturally occurring amino acid residues.

12. The method of claim 1 wherein the library entities are polymeric or oligomeric hydrazine aryl or alkyl carboxylic acids.

13. The method of claim 1 wherein the library entities are polymeric or oligomeric aminoxy aryl or alkyl carboxylic acids.

14. The method of claim 1 wherein the library entities have a molecular weight of more than 1000 Da.

15. The method of claim 14 wherein the library entities are oligomeric or polymeric compounds.

16. The method of claim 14 wherein the library entities are non-polymeric compounds.

17. The method of claim 1 wherein the library entities have a molecular weight of less than 1000 Da

18. The method of claim 17 wherein the library entities are oligomeric or polymeric compounds.

19. The method of claim 17 wherein the library entities are non-polymeric compounds.

20. The method of claim 1 wherein said entity tags are oligonucleotides.

21. The method of claim 20 wherein said oligonucleotide entity tags form a complex with oligonucleotide entity anti-tags.

22. The method of claim 21 wherein said oligonucleotides are selected from ribonucleic acids and deoxyribonucleic acids.

23. The method of claim 21 wherein said complex is a double stranded oligonucleotide selected from duplex DNA oligonucleotides and hybrid DNA/RNA oligonucleotides.

24. The method of claim 20 wherein the oligonucleotides have from 2 to 30 consecutive nucleotides linked by internucleoside linkers.

25. The method of claim 21 wherein the oligonucleotide entity tags and said oligonucleotide entity anti-tags have from 2 to 30 consecutive nucleotides linked by internucleoside linkers.

26. The method of claim 20, wherein the individual nucleotide residues of the oligonucleotide are composed of a nucleobase moiety and a backbone, wherein the backbone is composed of a sugar moiety and an internucleoside linker.

27. The method of claim 26 wherein the nucleobase moiety of the nucleotides of the oligonucleotide tags is selected from naturally occurring nucleobases.

28. The method of claim 26 wherein the nucleobase moiety of the nucleotides of the oligonucleotide tags is selected from adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine, N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diamino-purine, 5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine and inosine.

29. The method of claim 26 wherein the nucleobase moiety of the nucleotides of the oligonucleotide tags is selected from adenine, guanine, thymine, cytosine, 5-methylcytosine, and uracil.

30. The method of claim 26, wherein the backbone units of the oligonucleotide tags are selected from the group consisting of

31. The method of claim 26, wherein the sugar moiety of the backbone is a pentose.

32. The method of claim 26, wherein the pentose is selected from the group consisting of ribose, 2′

-deoxyribose, 2′

-O-methyl-ribose, 2′

-fluoro-ribose and 2′

-4′

-O-methylene-ribose (LNA).

33. The method of claim 26, wherein the internucleoside linker connects the 3′

end of a preceeding nucleotide monomer to the 5′

end of a succeeding nucleotide monomer, wherein the sugar moiety of the backbone is a pentose and wherein the internucleoside linker is selected from a phosphodiester linker, a phosphorothioate linker, a methylphosphonate linker, a phosphoramidate linker, a phosphotriester linker and a phosphodithioate linker.

34. The method of claim 26, wherein the nucleic acid monomers of the oligonucleotide include naturally occurring nucleosides of the DNA or RNA family connected through phosphodiester linkages.

35. The method of claim 34, wherein the members of the DNA family include deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine.

36. The method of claim 26, wherein the members of the RNA family include adenosine, guanosine, uridine, cytidine, and inosine.

37. The method of claim 1, wherein the chemical entity is associated with an entity tag through a covalent bond.

38. The method of claim 37, wherein the covalent bond is a cycloaddition product.

39. The method of claim 37, wherein the covalent bond is an alkylation product.

40. The method of claim 37, wherein the covalent bond is an arylation product.

41. The method of claim 37, wherein the covalent bond is an acylation product.

42. The method of claim 37, wherein the covalent bond is an amide bond.

43. The method of claim 37, wherein the covalent bond is a carboxylic ester bond.

44. The method of claim 37, wherein the covalent bond is a sulfonamide bond.

45. The method of claim 37, wherein the covalent bond is a disulfide bond.

46. The method of claim 37, wherein the covalent bond is an S-alkyl bond.

47. The method of claim 37, wherein the covalent bond is an NR-alkyl bond.

48. The method of claim 37, wherein the covalent bond is an O-alkyl bond.

49. The method of claim 37, wherein the covalent bond is an aryl-vinyl bond.

50. The method of claim 37, wherein the covalent bond is an alkyne-vinyl bond.

51. The method of claim 37, wherein the covalent bond is an oxime bond.

52. The method of claim 37, wherein the covalent bond is an imine bond.

53. The method of claim 37, wherein the covalent bond is a bicyclic product.

54. The method of claim 37, wherein the covalent bond is a trizole.

55. The method of claim 37, wherein the covalent bond is a hexene.

56. The method of claim 37, wherein the covalent bond is a 7-Oxa-bicyclo[2.2.1]hept-2-ene derivative.

57. The method of claim 37, wherein the covalent bond is a 7-Aza-bicyclo[2.2.1]hept-2-ene derivative.

58. The method of claim 37, wherein the covalent bond is 7-Methyl-7-aza-bicyclo[2.2.1]hept-2-ene.

59. The method of claim 1 wherein there are 10³-10⁶ entities in said library.

60. The method of claim 1 wherein there are 10³-10⁸ entities in said library.

61. The method of claim 1 wherein there are 10³-10¹⁰ entities in said library.

62. The method of claim 1 wherein there are 10⁵-10⁸ entities in said library.

63. The method of claim 1 wherein there are 10⁵-10¹⁰ entities in said library.

64. The method of claim 1 in which each fragment tag is directly attached to the fragment it identifies, prior to ligation of the fragments to form the chemical entity and prior to the ligation of the fragment tags to form the entity tag.

65. The method of claim 64 in which the fragments are ligated to form the chemical entity prior to the ligation of the attached fragment tags to form the entity tag.

66. The method of claim 64 in which the fragment tags are ligated to form the entity tag prior to the ligation of the fragments to form the chemical entity.

67. In a chemical screening method, which comprises screening a combinatorial library comprising a plurality of structurally distinct chemical entities, said screening comprising (1) the substantially simultaneous determination of whether any of said library entities have a predetermined binding activity or biological activity, and, if at least one library entity has such activity, (2) the determination of the chemical structure of the at least one library entity having such activity,

wherein, for purposes of determination of the chemical structure of the library entity, each chemical entity is tagged with an entity tag identifying the structure of the chemical entity,

wherein each chemical entity comprises a plurality of fragments,

wherein the entity tag consists essentially of a plurality of fragment tags, each fragment tag identifying one of the fragments constituting a fragment of the chemical entity tagged by said entity tag,

the improvement comprising

ligating at least one pair of fragment tags by enzyme-catalysed ligation.