Multi-step synthesis of templated molecules

• US 10,730,906 B2
• Filed: 07/30/2003
• Issued: 08/04/2020
• Est. Priority Date: 08/01/2002
• Status: Active Grant

• Current Assignee: Nuevolution A/S (Amgen Inc.)
• Original Assignee: Nuevolutions A/S
• Inventors: Pedersen, Henrik
• Primary Examiner(s): Vivlemore, Tracy
• Assistant Examiner(s): Kaup, Sahana S
• Application Number:
  US10/523,006
• Publication Number:
  US 20060121470A1
• Time in Patent Office: 6,215 Days
• Field of Search: None
• 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

Disclosed is a method for the manufacture of a library of complexes. The complexes comprise templated molecules attached to the template which directed the synthesis thereof. The templated molecules are produced in a step-by-step fashion which provides for a high local concentration of reactive groups involved in the formation of connections between the individual components of the template molecule.

Claims

1. A library comprising different complexes, said complexes comprising a first entity and a second entity, wherein

the first entity comprises a double stranded oligonucleotide identifier comprising single oligonucleotide strands covalently linked by a first linker, each single strand containing from 12 to 400 nucleic acid monomers, wherein at least one nucleic acid monomer of each single strand is a deoxyribonucleotide,

wherein the second entity comprises a small non-peptide molecule and a second linker, the second linker being distinct from the first linker and covalently linking the first entity to the small-non-peptide molecule of the second entity,

wherein the small non-peptide molecule of the second entity can be identified by each and both strands of the double stranded oligonucleotide identifier of the first entity, and

wherein the small non-peptide molecule is covalently linked to the double stranded identifier oligonucleotide via an anchorage point located at a terminal region of one of the single strands.

2. The library according to claim 1, wherein the small non-peptide molecule of the complexes of the library is selected from the group consisting of monofunctional, difunctional,trifunctional and oligofunctional open-chain hydrocarbons;

monofunctional, difunctional,trifunctional and oligofunctional non-aromatic carbocycles;

monocyclic, bicyclic, tricyclic and polycyclic hydrocarbons;

bridged polycyclic hydrocarbons;

monofunctional, difunctional, trifunctional, and oligofunctional non-aromatic heterocycles;

monocyclic, bicyclic, tricyclic, and polycyclic heterocycles, bridged polycyclic heterocycles;

monofunctional, difunctional,trifunctional and oligofunctional aromatic carbocycles;

monocyclic, bicyclic, tricyclic, and polycyclic aromatic carbocycles; and

monofunctional, difunctional,trifunctional and oligofunctional aromatic heterocycles.

3. The library of claim 1, wherein the number of complexes is from 2 to 10¹⁸.

4. The library of claim 1, wherein the individual nucleic acid monomers of the covalently linked oligonucleotide identifier comprise a nucleobase moiety and a sugar moiety and an internucleoside linker.

5. The library of claim 4, wherein the nucleobase moiety of the nucleic acid monomers is a natural nucleobase moiety.

6. The library of claim 5, wherein the nucleobase moieties are selected from the group consisting of deoxyadenosine, deoxyguanosine, deoxythymidine, deoxycytidine, adenosine, guanosine, uridine, cytidine and inosine.

7. The library of claim 4, wherein the sugar moiety of the nucleic acid monomers is a pentose.

8. The library of claim 7, wherein the pentose is selected from the group consisting of ribose, 2′

-deoxyribose, 2′

-O-methyl-ribose, 2′

-flouro-ribose, and 2′

-4′

-O-methylene-ribose.

9. The library of claim 1, wherein the internucleoside linker linking the individual nucleic acid monomers is a phosphodiester linker.

10. The library according to claim 1, wherein the first and/or second linker is selected from the group consisting of carbohydrides, substituted carbohydrides, vinyl, polyvinyl, substituted polyvinyl, acetylene, polyacetylene, aryl /hetaryl, polyaryl/hetaryl and substituted polyaryl/polyhetaryl, ethers, polyethers, amines, polyamines, substituted polyamines;

double stranded, single stranded or partially double stranded natural and unnatural polynucleotides, substituted double stranded, single stranded or partially double stranded natural and unnatural polynucleotides, polyamides, natural and un-natural polypeptides, substituted polyamides, and substituted natural and unnatural polypeptides.

11. The library according to claim 1, wherein said first and/or second linker comprises a polynucleotide linker.

12. The library according to claim 1, wherein said first and/or second linker comprises a polyether linker.

13. The library according to claim 12 wherein said first and/or second linker comprises a polyethyleneglycol linker.

14. The library according to claim 12 wherein said first and/or second linker comprises a substituted polyether linker.

15. The library of claim 4, wherein the nucleobase moiety of the nucleic acid monomers is selected from the group consisting of 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, pseudo-isocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, and inosine.

16. The library of claim 1, wherein the first linker of the double stranded identifier oligonucleotide comprises a hair-pin loop of nucleotides covalently linking the two strands of the double stranded identifier oligonucleotide at one end of the strands.

17. The library of claim 1, wherein the small non-peptide molecule is covalently linked to the double stranded identifier oligonucleotide via an anchorage point located at a first terminal region of one of the single strands, and wherein the first linker comprises a hair-pin loop of nucleotides covalently linking the two strands of the double stranded identifier oligonucleotide at a different, second terminal region of the single strands.

18. A method for the enrichment of the library according to claim 1 for small non-peptide molecules having a predetermined activity or functionality, said method comprising the steps of:

(i) subjecting said library one or more times to an enrichment condition, and

(ii) obtaining an enriched library having a higher relative amount of small non-peptide molecules having said predetermined activity or functionality.

19. The method of claim 18, wherein the oligonucleotide identifiers of the enriched complexes are amplified.

20. The method of claim 18, wherein the small non-peptide molecules of the enriched library are identified by sequencing the oligonucleotide identifier.

21. The method of claim 18, wherein the enrichment condition is an affinity of the small non-peptide molecules of the library of claim 1 for a target molecule or target entity.

22. The library according to claim 11, wherein each of the first and the second linker comprises a polynucleotide linker.

23. The library according to claim 12, wherein each of the first and the second linker comprises a polyether linker.

24. The library according to claim 13, wherein each of the first and the second linker comprises a polyethyleneglycol linker.

43. The library according to claim 1, wherein each nucleic acid monomer present in the oligonucleotide identifier comprises a naturally occurring nucleobase and a backbone moiety.

44. The library according to claim 1, wherein the nucleobases of the nucleic acid monomers of the oligonucleotide identifier are selected from the groupd consisting of naturally occurring nucleobases and non-naturally occurring nucleobases, wherein the nucleobases are connected by backbone moieties comprising a pentose sugar moiety and an internucleoside linker.

45. The library according to claim 44, wherein the nucleobases of the nucleic acid monomers of the identifier are selected from the group consisting of purine and pyrimidine hetero-cycles, including heterocyclic analogues and tautomers thereof.

46. The library according to claim 45, wherein the nucleobases of the nucleic acid monomers are selected from the group consisting of

adenine, 8-oxo-N⁶-methyladenine;

guanine, isoguanine, 7-deazaguanine;

cytosine, isocytosine, pseudoisocytosine, N⁴,N⁴-ethanocytosine, 5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine;

thymine;

uracil, 5-bromouracil, 5-fluorouracil;

inosine;

purine, diaminopurine, N⁶,N⁶-ethano-2,6-diamino-purine;

xanthine, 7-deazaxanthine;

pyrimidine and 2-hydroxy-5-methyl-4-triazolopyridine;

including heterocyclic analogues and tautomers thereof.

47. The library according to claim 44, wherein each backbone moiety is independently selected from the group consisting of

48. The library according to claim 1, wherein each nucleic acid monomer present in the oligonucleotide identifier is composed of a nucleobase and a backbone moiety, wherein each nucleobase is selected from the group consisting of naturally occurring nucleobases and non-naturally occurring nucleobases, and wherein each backbone moiety comprises a pentose sugar moiety and an internucleoside linker.

49. The library according to claim 48, wherein the pentose sugar moiety is selected from the group consisting of ribose, 2′

-deoxyribose, 2′

-O-methyl-ribose, 2′

-flouro-ribose, and 2′

-4′

-O-methylene-ribose (LNA), and wherein each nucleobase is attached to the 1′

position of each pentose sugar moiety.

50. The library according to claim 48, wherein each internucleoside linker is connecting the 3′

end of a preceding pentose monomer to a 5′

end of a succeeding pentose monomer in the identifier oligonucleotide.

51. The library according to claim 50, wherein each internucleoside linker is independently selected from the group of consisting of a phosphodiester linker, a phosphorothioate linker, a methylphosphonate linker, a phosphoramidate linker, a phosphotriester linker, a phosphodithioate linker, and a non-phosphorous-containing linker.

52. The library according to claim 1, wherein the olgionucleotide identifier comprises nucleic acid monomers selected from the group consisting of nucleosides consisting of deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine, wherein said nucleosides are connected through phosphodiester linkages.

53. The library according to claim 1, wherein the oligonucleotide identifier comprises nucleic acid monomers selected from the group consisting of nucleosides consisting of adenosine, guanosine, uridine, cytidine, and inosine, wherein said nucleosides are connected through phosphodiester linkages.

54. The library according to claim 1, wherein the oligonucleotide identifier comprises nucleic acid monomers selected from a first group consisting of nucleosides consisting of deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine, as well as nucleosides selected from a second group consisting of nucleosides consisting of adenosine, guanosine, uridine, cytidine, and inosine, wherein said nucleosides are connected through phosphodiester linkages.

55. The library according to claim 1, wherein the second linker is a polyethylene glycol (PEG) linker.

56. The library according to claim 55, wherein the small non-peptide small non-peptide molecules of the library are selected from the group consisting of

monofunctional, difunctional, trifunctional and oligofunctional, open-chain hydrocarbons, monocyclic, bicyclic, tricyclic and polycyclic hydrocarbons, bridged polycyclic hydrocarbons;

monofunctional, difunctional trifunctional and oligofunctional, non-aromatic carbocycles, monofunctional, difunctional, trifunctional and oligofunctional, aromatic carbocycles, monocyclic, bicyclic, tricyclic and polycyclic, aromatic carbocycles;

monofunctional, difunctional, trifunctional and oligofunctional, non-aromatic heterocycles, monofunctional, difunctional, trifunctional and oligofunctional, aromatic heterocycles monocyclic, bicyclic, tricyclic and polycyclic heterocycles, and bridged polycyclic heterocycles.

25. A library comprising different complexes, wherein each complex comprises

i) a first entity comprising a double stranded oligonucleotide identifier comprising single oligonucleotide strands covalently linked by a first linker,

ii) a second entity comprising a small non-peptide molecule, and

iii) a second linker covalently linking the first entity to the small non-peptide molecule of the second entity,

wherein the small non-peptide molecule of the second entity can be identified by each and both of the single strands of the double stranded oligonucleotide identifier of the first entity, wherein the small non-peptide molecule is covalently linked to the double stranded identifier oligonucleotide via an anchorage point located at a terminal region of one of the single strands.

26. The library according to claim 25, wherein the small non-peptide molecule of the complexes of the library is selected from the group consisting of

monofunctional, difunctional, trifunctional and oligofunctional open-chain hydrocarbons;

monofunctional, difunctional, trifunctional and oligofunctional non-aromatic carbocycles;

monocyclic, bicyclic, tricyclic and polycyclic hydrocarbons;

bridged polycyclic hydrocarbons;

monofunctional, difunctional, trifunctional, and oligofunctional non-aromatic heterocycles;

monocyclic, bicyclic, tricyclic, and polycyclic heterocycles, bridged polycyclic heterocycles;

monofunctional, difunctional, trifunctional and oligofunctional aromatic carbocycles;

monocyclic, bicyclic, tricyclic, and polycyclic aromatic carbocycles; and

monofunctional, difunctional, trifunctional and oligofunctional aromatic heterocycles.

27. The library of claim 25, wherein the number of complexes is from 2 to 10¹⁸.

28. The library of claim 25, wherein the individual nucleic acid monomers of the covalently linked oligonucleotide identifier comprise a nucleobase moiety and a sugar moiety and an internucleoside linker.

29. The library according to claim 25, wherein the first and/or second linker is selected from the group consisting of carbohydrides, substituted carbohydrides, vinyl, polyvinyl, substituted polyvinyl, acetylene, polyacetylene, aryl /hetaryl, polyaryl/hetaryl and substituted polyaryl/polyhetaryl, ethers, polyethers, amines, polyamines, substituted polyamines;

double stranded, single stranded or partially double stranded natural and unnatural polynucleotides, substituted double stranded, single stranded or partially double stranded natural and unnatural polynucleotides, polyamides, natural and un-natural polypeptides, substituted polyamides, and substituted natural and unnatural polypeptides.

30. The library according to claim 25, wherein said first and/or second linker comprises a polynucleotide linker.

31. The library according to claim 25, wherein said first and/or second linker comprises a polyether linker.

32. The library according to claim 25 wherein said first and/or second linker comprises a polyethyleneglycol linker.

33. The library according to claim 25 wherein said first and/or second linker comprises a substituted polyether linker.

34. The library according to claim 30, wherein each of the first and the second linker comprises a polynucleotide linker.

35. The library according to claim 31, wherein each of the first and the second linker comprises a polyether linker.

36. The library according to claim 32 wherein each of the first and the second linker comprises a polyethyleneglycol linker.

37. The library of claim 25, wherein the first linker of the double stranded identifier oligonucleotide comprises a hair-pin loop of nucleotides covalently linking the two strands of the double stranded identifier oligonucleotide at one end of the strands.

38. The library of claim 25, wherein the small non-peptide molecule is covalently linked to the double stranded identifier oligonucleotide via an anchorage point located at a first terminal region of one of the single strands, and wherein the first linker comprises a hair-pin loop of nucleotides covalently linking the two strands of the double stranded identifier oligonucleotide at a different, second terminal region of the single strands.

39. A method for the enrichment of the library according to claim 25 for small non-peptide molecules having a predetermined activity or functionality, said method comprising the steps of:

(i) subjecting said library one or more times to an enrichment condition, and

(ii) obtaining an enriched library having a higher relative amount of small non-peptide molecules having said predetermined activity or functionality.

40. The method of claim 39, wherein the oligonucleotide identifiers of the enriched complexes are amplified.

41. The method of claim 39, wherein the small non-peptide molecules of the enriched library are identified by sequencing the oligonucleotide identifier.

42. The method of claim 39, wherein the enrichment condition is an affinity of the small non-peptide molecules of the library of claim 25 for a target molecule or target entity.

57. The library according to claim 25, wherein each nucleic acid monomer present in the oligonucleotide identifier comprises a naturally occurring nucleobase and a backbone moiety.

58. The library according to claim 25, wherein the nucleobases of the nucleic acid monomers of the oligonucleotide identifier are selected from the group consisting of naturally occurring nucleobases and non-naturally occurring nucleobases, wherein the nucleobases are connected by backbone moieties comprising a pentose sugar moiety and an internucleoside linker.

59. The library according to claim 58, wherein the nucleobases of the nucleic acid monomers of the oligonucleotide identifier are selected from the group consisting of purine and pyrimidine hetero-cycles, including heterocyclic analogues and tautomers thereof.

60. The library according to claim 59, wherein the nucleobases of the nucleic acid monomers are selected from the group consisting of

adenine, 8-oxo-N⁶-methyladenine;

guanine, isoguanine, 7-deazaguanine;

cytosine, isocytosine, pseudoisocytosine, N⁴,N⁴-ethanocytosine, 5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine;

thymine;

uracil, 5-bromouracil, 5-fluorouracil;

inosine;

purine, diaminopurine, N⁶,N⁶-ethano-2,6-diamino-purine;

xanthine, 7-deazaxanthine;

pyrimidine and 2-hydroxy-5-methyl-4-triazolopyridine;

including heterocyclic analogues and tautomers thereof.

61. The library according to claim 58, wherein each backbone moiety is independently selected from the group consisting of

62. The library according to claim 25, wherein each nucleic acid monomer present in the oligonucleotide identifier is composed of a nucleobase and a backbone moiety, wherein each nucleobase is selected from the group consisting of naturally occurring nucleobases and non-naturally occurring nucleobases, and wherein each backbone moiety comprises a pentose sugar moiety and an internucleoside linker.

63. The library according to claim 62, wherein the pentose sugar moiety is selected from the group consisting of ribose, 2′

-deoxyribose, 2′

-O-methyl-ribose, 2′

-flouro-ribose, and 2′

-4′

-O-methylene-ribose (LNA), and wherein each nucleobase is attached to the 1′

position of each pentose sugar moiety.

64. The library according to claim 62, wherein each internucleoside linker is connecting the 3′

end of a preceding pentose monomer to a 5′

end of a succeeding pentose monomer in the identifier oligonucleotide.

65. The library according to claim 64, wherein each internucleoside linker is independently selected from the group of consisting of a phosphodiester linker, a phosphorothioate linker, a methylphosphonate linker, a phosphoramidate linker, a phosphotriester linker, a phosphodithioate linker, and a non-phosphorous-containing linker.

66. The library according to claim 25, wherein the oligonucleotide identifier comprises nucleic acid monomers selected from the group consisting of nucleosides consisting of deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine, wherein said nucleosides are connected through phosphodiester linkages.

67. The library according to claim 25, wherein the oligonucleotide identifier comprises nucleic acid monomers selected from the group consisting of nucleosides consisting of adenosine, guanosine, uridine, cytidine, and inosine, wherein said nucleosides are connected through phosphodiester linkages.

68. The library according to claim 25, wherein the oligonucleotide identifier comprises nucleic acid monomers selected from a first group consisting of nucleosides consisting of deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine, as well as nucleosides selected from a second group of nucleosides consisting of adenosine, guanosine, uridine, cytidine, and inosine, wherein said nucleosides are connected through phosphodiester linkages.

69. The library according to claim 25, wherein the second linker is a polyethylene glycol (PEG) linker.

70. The library according to claim 69, wherein the small non-peptide molecules of the library are selected from the group consisting of

monofunctional, difunctional, trifunctional and oligofunctional, open-chain hydrocarbons, monocyclic, bicyclic, tricyclic and polycyclic hydrocarbons, bridged polycyclic hydrocarbons;

monofunctional, difunctional trifunctional and oligofunctional, non-aromatic carbocycles, monofunctional, difunctional, trifunctional and oligofunctional, aromatic carbocycles, monocyclic, bicyclic, tricyclic and polycyclic, aromatic carbocycles;

monofunctional, difunctional, trifunctional and oligofunctional, non-aromatic heterocycles, monofunctional, difunctional, trifunctional and oligofunctional, aromatic heterocycles monocyclic, bicyclic, tricyclic and polycyclic heterocycles, and bridged polycyclic heterocycles.

71. A library comprising different complexes, wherein each complex comprises

i) a first entity comprising a double stranded oligonucleotide identifier comprising single oligonucleotide strands covalently linked by a first linker, wherein the first linker links oligonucleotide strand 3′

and 5′

ends,

ii) a second entity comprising a small non-peptide molecule, and

iii) a second linker covalently linking the first entity to the second entity,

wherein the small non-peptide molecule of the second entity can be identified by each and both of the single strands of the double stranded oligonucleotide identifier,

wherein the first entity is covalently linked to the second entity at or near the 3′

or 5′

end of a single strand of the double stranded oligonucleotide identifier.

72. The library of claim 71, wherein the number of complexes is from 2 to 10¹⁸.

73. The library according to claim 72, wherein the small non-peptide molecule of the complexes of the library is selected from the group consisting of monofunctional, difunctional,trifunctional and oligofunctional open-chain hydrocarbons;

monofunctional, difunctional,trifunctional and oligofunctional non-aromatic carbocycles;

monocyclic, bicyclic, tricyclic and polycyclic hydrocarbons;

bridged polycyclic hydrocarbons;

monofunctional, difunctional, trifunctional, and oligofunctional non-aromatic heterocycles;

monocyclic, bicyclic, tricyclic, and polycyclic heterocycles, bridged polycyclic heterocycles;

monofunctional, difunctional,trifunctional and oligofunctional aromatic carbocycles;

monocyclic, bicyclic, tricyclic, and polycyclic aromatic carbocycles; and

monofunctional, difunctional,trifunctional and oligofunctional aromatic heterocycles.

74. The library according to claim 73, wherein the first and/or second linker is selected from the group consisting of carbohydrides, substituted carbohydrides, vinyl, polyvinyl, substituted polyvinyl, acetylene, polyacetylene, aryl /hetaryl, polyaryl/hetaryl and substituted polyaryl/polyhetaryl, ethers, polyethers, amines, polyamines, substituted polyamines;

double stranded, single stranded or partially double stranded natural and unnatural polynucleotides, substituted double stranded, single stranded or partially double stranded natural and unnatural polynucleotides, polyamides, natural and un-natural polypeptides, substituted polyamides, and substituted natural and unnatural polypeptides.

75. The library according to claim 74, wherein said first and/or second linker comprises a polynucleotide linker.

76. The library according to claim 74, wherein said first and/or second linker comprises an optionally substituted polyether linker.

77. The library according to claim 74, wherein said first and/or second linker comprises a polyethyleneglycol linker.

78. The library of claim 75, wherein the first linker comprises a hair-pin loop of nucleotides.

79. The library of claim 78, wherein the second entity is covalently linked to the first entity at or near the 5′

or 3′

end of a single strand of double stranded identifier oligonucleotide, and wherein the first linker comprises a hair-pin loop comprising nucleotides covalently linking the two strands of the double stranded identifier oligonucleotide at the opposite end of the single strand.

80. The library according to claim 76, wherein the second linker comprises an optionally substituted polyether linker.

81. The library according to claim 77, wherein the second linker comprises a polyethylene glycol.

82. The library according to claim 80, wherein the polyether linker is not substituted.

83. The library according to claim 71, wherein each nucleic acid monomer present in the oligonucleotide identifier is composed of a nucleobase and a backbone moiety, wherein each nucleobase is selected from the group consisting of naturally occurring nucleobases and non-naturally occurring nucleobases, and wherein each backbone moiety comprises a pentose sugar moiety and an internucleoside linker.

84. The library according to claim 83, wherein the pentose sugar moiety is selected from the group consisting of ribose, 2′

-deoxyribose, 2′

-O-methyl-ribose, 2′

-flouro-ribose, and 2′

-4′

-O-methylene-ribose (LNA).

85. The library according to claim 83, wherein each internucleoside linker connects the 3′

end of a preceding pentose monomer to a 5′

end of a succeeding pentose monomer, and wherein each internucleoside linker is independently selected from the group consisting of a phosphodiester linker, a phosphorothioate linker, a methylphosphonate linker, a phosphoramidate linker, a phosphotriester linker, a phosphodithioate linker and a non-phosphorous-containing linker.

86. The library according to claim 85, wherein at least one internucleoside linker is a non-phosphorous-containing linker.

87. A library comprising different complexes, wherein each complex comprises

i) a first entity comprising a double stranded oligonucleotide identifier comprising single oligonucleotide strands covalently linked by a terminally located hair-pin loop comprising nucleotides, wherein the hairpin loop covalently links oligonucleotide strand 3′

and 5′

ends.

ii) a second entity comprising a molecule selected from the group consisting of monofunctional, difunctional, trifunctional and oligofunctional open-chain hydrocarbons;

monofunctional, difunctional, trifunctional and oligofunctional non-aromatic carbocycles;

monocyclic, bicyclic, tricyclic and polycyclic hydrocarbons;

bridged polycyclic hydrocarbons;

monofunctional, difunctional, trifunctional, and oligofunctional non-aromatic heterocycles;

monocyclic, bicyclic, tricyclic, and polycyclic heterocycles, bridged polycyclic heterocycles;

monofunctional, difunctional, trifunctional and oligofunctional aromatic carbocycles;

monocyclic, bicyclic, tricyclic, and polycyclic aromatic carbocycles; and

monofunctional, difunctional, trifunctional and oligofunctional aromatic heterocycles,

iii) a linker covalently linking the first entity and the second entity,

wherein the molecule of the second entity can be identified by each and both of the single strands of the double stranded oligonucleotide identifier,

wherein the first entity is covalently linked to the second entity at a terminal location of the first entity.

88. The library according to claim 87, wherein the linker covalently linking the first entity and the second entity is selected from the group consisting of carbohydrides, substituted carbohydrides, vinyl, polyvinyl, substituted polyvinyl, acetylene, polyacetylene, aryl/hetaryl, polyaryl/hetaryl and substituted polyaryl/polyhetaryl, ethers, polyethers, amines, polyamines, substituted polyamines;

double stranded, single stranded or partially double stranded natural and unnatural polynucleotides, substituted double stranded, single stranded or partially double stranded natural and unnatural polynucleotides, polyamides, natural and un-natural polypeptides, substituted polyamides, and substituted natural and unnatural polypeptides.

89. The library according to claim 87, wherein the linker covalently linking the first entity and the second entity comprises an optionally substituted polyether linker.

90. The library according to claim 87, wherein the linker covalently linking the first entity and the second entity comprises a polyethylene glycol.

91. The library according to claim 89, wherein the polyether is not substituted.

92. The library according to claim 87, wherein each nucleic acid monomer present in the oligonucleotide identifier is composed of a nucleobase and a backbone moiety, wherein each nucleobase is selected from the group consisting of naturally occurring nucleobases and non-naturally occurring nucleobases, and wherein each backbone moiety comprises a pentose sugar moiety and an internucleoside linker.

93. The library according to claim 92, wherein the pentose sugar moiety is selected from the group consisting of ribose, 2′

-deoxyribose, 2′

-O-methyl-ribose, 2′

-flouro-ribose, and 2′

-4′

-O-methylene-ribose (LNA).

94. The library according to claim 92, wherein each internucleoside linker connects the 3′

end of a preceding pentose monomer to a 5′

end of a succeeding pentose monomer, and wherein each internucleoside linker is independently selected from the group consisting of a phosphodiester linker, a phosphorothioate linker, a methylphosphonate linker, a phosphoramidate linker, a phosphotriester linker, a phosphodithioate linker, and a non-phosphorous-containing linker.

95. The library according to claim 94, wherein at least one internucleoside linker is a non-phosphorous-containing linker.

96. The library according to claim 95, wherein further internucleoside linkers of the identifier oligonucleotide are independently selected from the group of consisting of a phosphodiester linker, a phosphorothioate linker, a methylphosphonate linker, a phosphoramidate linker, a phosphotriester linker and a phosphodithioate linker.

97. The method of claim 87, wherein the terminal location of the first entity is a terminal region of either of the covalently linked single strands of the double stranded oligonucleotide identifier.

98. The library according to claim 87, wherein the second entity is covalently linked to the first entity at a terminal region of a single strand of the double stranded identifier oligonucleotide, and wherein the hair-pin loop covalently links the two strands of the double stranded identifier oligonucleotide at the opposite end of the single strand.