Quasirandom structure and function guided synthesis methods

• US 9,121,110 B2
• Filed: 12/19/2003
• Issued: 09/01/2015
• Est. Priority Date: 12/19/2002
• Status: Active Grant

• Current Assignee: Nuevolution A/S (Amgen Inc.)
• Original Assignee: Nuevolution A/S (Amgen Inc.)
• Inventors: Gouliaev, Alex Haahr, Holtmann, Anette, Pedersen, Henrik, Franch, Thomas
• Primary Examiner(s): Crow, Robert T
• Assistant Examiner(s): Dauner, Joseph G
• Application Number:
  US10/539,288
• Publication Number:
  US 20080193983A1
• Time in Patent Office: 4,274 Days
• Field of Search: None
• US Class Current: 1/1
• 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 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/04 using dynamic combinatorial chemistry techniques
• CPC Class Codes: C40B 50/08 Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: Y10T 436/145555 Hetero-N

Abstract

The present invention is directed to the synthesis of molecules guided by connector polynucleotides (CPNs) capable of hybridizing to complementary connector polynucleotides (CCPNs) harboring at least one functional entity comprising at least one reactive group. At least one of the CCPNs is capable of hybridizing to at least two CPNs. Each CPN will “call” for one or more CCPNs capable of hybridization to the CPN. Following the formation of a supramolecular hybridization complex comprising a plurality of CPNs and a plurality of CCPNs, the reaction of reactive groups results in the formation of a molecule comprising covalently linked functional entities. The formation of the molecule involves the transfer of functional entities from one or more “donor CCPNs” to at least one “acceptor CCPN” with which the transferred functional entities were not associated prior to the transfer.

Claims

1. A method for synthesising at least one bifunctional molecule comprising a molecule part linked to a nucleic acid part, comprising the steps of

i) providing at least 2 connector polynucleotides each capable of hybridizing to at least 1 complementary connector polynucleotide,

ii) providing at least 2 complementary connector polynucleotides each capable of hybridizing to at least 1 connector polynucleotide,

iii) hybridizing at least 2 complementary connector polynucleotides to at least 2 connector polynucleotides to form the nucleic acid part as a hybridization complex, wherein at least 2 of said complementary connector polynucleotides comprise at least 1 functional entity, wherein at least 1 of said complementary connector polynucleotides hybridizes to at least 2 connector polynucleotides, and with the proviso that no single complementary connector or connector polynucleotide of the hybridization complex hybridizes to all of the remaining complementary connector or connector polynucleotides of the hybridization complex;

iv) covalently reacting at least 2 functional entities, wherein the covalent reaction of the functional entities results in the formation of a molecule of the molecule part, wherein the functional entities are provided by separate complementary connector polynucleotides,

thereby providing the at least one bifunctional molecule wherein the nucleic acid part of the bifunctional molecule comprises nucleotide sequences that identify the functional entities that covalently reacted to form the molecule comprised within the bifunctional molecule.

2. The method of claim 1, wherein step iv) comprises reacting at least 3 or functional entities.

3. The method of claim 1, wherein step iii) comprises

iii) hybridizing at least 3 complementary connector polynucleotides to at least 2 connector polynucleotides, wherein at least 3 of said complementary connector polynucleotides comprise at least 1 functional entity, wherein at least 1 of said complementary connector polynucleotides hybridizes to at least 2 connector polynucleotides, and

wherein step iv) comprises reacting at least 3 functional entities, wherein the reaction of said functional entities results in the formation of the molecule by covalently linking at least 3 functional entities provided by separate complementary connector polynucleotides.

4. The method of claim 3, wherein in step iv), at least 4 functional entities are reacted.

5. The method of claim 1, wherein step iii) comprises

hybridizing at least 4 complementary connector polynucleotides to at least 2 connector polynucleotides, wherein at least 4 of said complementary connector polynucleotides comprise at least 1 functional entity, wherein at least 1 of said complementary connector polynucleotides hybridizes to at least 2 connector polynucleotides, and wherein step iv) comprises

reacting at least 4 functional entities,

wherein the reaction of said functional entities results in the formation of the molecule by covalently linking at least 4 functional entities provided by separate complementary connector polynucleotides.

6. The method of claim 5, wherein in step iv), at least 5 functional entities are reacted.

7. The method of claim 1, wherein step iii) comprises

hybridizing at least 5 complementary connector polynucleotides to at least 2 connector polynucleotides, wherein at least 5 of said complementary connector polynucleotide comprise at least 1 functional entity, wherein at least 1 of said complementary connector polynucleotides hybridizes to at least 2 connector polynucleotides,

and wherein step iv) comprises

reacting at least 5 functional entities, wherein the reaction of said functional entities results in the formation of the molecule by covalently linking the functional entities provided by separate complementary connector polynucleotides.

8. The method of claim 7, wherein in step iv), at least 6 functional entities are reacted.

9. The method of claim 1, wherein the molecule comprising covalently linked functional entities is linked to the polynucleotide part of a complementary connector polynucleotide.

10. The method of claim 1 comprising the further step of cleaving at least one linker linking the molecule comprising covalently linked functional entities to the polynucleotide part of a complementary connector polynucleotide.

11. The method of claim 10, wherein all linkers but 1 linker are cleaved, and wherein the linker not cleaved links the molecule to the polynucleotide part of a complementary connector polynucleotide.

12. The method of claim 1, wherein complementary connector polynucleotides hybridized to connector polynucleotides are not linked by covalent bonds when reaction step iv) has been carried out, and/or wherein the polynucleotide part of different connector polynucleotides and/or different complementary connector polynucleotides are not covalently linked prior to the reactions of step iv).

13. The method of claim 12 comprising the further step of linking the complementary connector polynucleotides, optionally preceded by initially performing a polynucleotide extension reaction resulting in individual complementary connector polynucleotides being linked together by covalent bonds.

14. The method of claim 1, wherein connector polynucleotides hybridized to complementary connector polynucleotides are not linked by covalent bonds when reaction step iv) has been carried out, and/or wherein the polynucleotide part of different connector polynucleotides and/or different complementary connector polynucleotides are not covalently linked prior to the reactions of step iv).

15. The method of claim 14 comprising the further step of linking the connector polynucleotides, optionally preceded by performing a polynucleotide extension reaction resulting in individual connector polynucleotides being linked together by covalent bonds.

16. The method of claim 1 comprising the further steps of

a) linking the complementary connector polynucleotides, optionally preceded by performing a polynucleotide extension reaction resulting in individual complementary connector polynucleotides being linked together by covalent bonds, and

b) linking the connector polynucleotides, optionally preceded by performing a polynucleotide extension reaction resulting in individual connector polynucleotides being linked together by covalent bonds.

17. The method of claim 1, wherein the method does not involve ribosome mediated translation.

18. The method of claim 1 further comprising the step of hybridizing at least 1 further connector polynucleotide to at least 1 complementary connector polynucleotide, hybridized to at least 1 connector polynucleotide, of the hybridisation complex of step iii).

19. The method of claim 18, wherein the further connector polynucleotide is selected from the group consisting of

connector polynucleotides comprising at least 1 functional entity,

and

connector polynucleotides comprising at least 1 spacer region.

20. The method of claim 18, wherein the step of hybridizing at least 1 further connector polynucleotide is repeated at least once.

21. The method of claim 1 further comprising the step of hybridizing at least 1 further complementary connector polynucleotide selected from the group consisting of

complementary connector polynucleotides comprising at least 1 functional entity,

and

complementary connector polynucleotides comprising at least 1 spacer region,

to the hybridisation complex of step iii), or to at least 1 further connector polynucleotide of said hybridisation complex.

22. The method of claim 21, wherein the step of hybridising at least one further complementary connector polynucleotide is repeated at least once.

23. The method of claim 1, wherein at least n connector polynucleotides and at least n−

1 complementary connector polynucleotides are provided, n being an integer of from 3 to 6, and wherein each complementary connector polynucleotide hybridizes to at least 2 connector polynucleotides.

24. The method of claim 23, wherein n is 3 or 4.

25. The method of claim 1, wherein at least n connector polynucleotides and at least n complementary connector polynucleotides are provided, n being an integer of from 3 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

26. The method of claim 25, wherein n complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

27. The method of claim 25, wherein n is 3 or 4.

28. The method of claim 1, wherein at least n connector polynucleotides and at least n+1 complementary connector polynucleotides are provided, n being an integer of from 3 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

29. The method of claim 28, wherein n complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

30. The method of claim 28, wherein n is 3 or 4.

31. The method of claim 1, wherein at least n connector polynucleotides and at least n+2 complementary connector polynucleotides are provided, n being an integer of from 3 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

32. The method of claim 31, wherein n complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

33. The method of claim 31, wherein n is 3 or 4.

34. The method of claim 1, wherein at least n connector polynucleotides and at least n+3 complementary connector polynucleotides are provided, n being an integer of from 3 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

35. The method of claim 34, wherein n complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

36. The method of claim 34, wherein n is 3 or 4.

37. The method of claim 1, wherein at least n connector polynucleotides and at least n+4 complementary connector polynucleotides are provided, n being an integer of from 3 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

38. The method of claim 37, wherein n complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

39. The method of claim 37, wherein n is 3 or 4.

40. The method of claim 1, wherein said at least 2 connector polynucleotides comprises branched connector polynucleotides, wherein at least n branched connector polynucleotides and at least n complementary connector polynucleotides are provided, n being an integer of from 2 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 branched connector polynucleotides.

41. The method of claim 40, wherein at least n+1 complementary connector polynucleotides are provided.

42. The method of claim 40, wherein at least n complementary connector polynucleotides hybridize to at least 2 branched connector polynucleotides.

43. The method of claim 42, wherein at least n+1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

44. The method of claim 40, wherein n is 3 or 4.

45. The method of claim 1 comprising the further step of repeating, for different connector polynucleotides and different complementary connector polynucleotides, the steps iii) and iv) at least once, wherein the different complementary connector polynucleotides are hybridised, in each repeated step iii), to the hybridisation complex having been generated in the previous steps of the method, and wherein different functional entities are linked in each repeated step iv).

46. The method of claim 1, wherein at least 1 functional entity of a complementary connector polynucleotide reacts with the functional entities of at least 2 other complementary connector polynucleotides.

47. The method of claim 1, wherein at least 1 functional entity of a complementary connector polynucleotide reacts with the functional entities of at least 3 other complementary connector polynucleotides.

48. The method of claim 1, wherein said at least 2 complementary connector polynucleotides comprise at least 2 complementary connector polynucleotides which are non-identical.

49. The method of claim 1, wherein said at least 2 complementary connector polynucleotides comprise at least 2 branched complementary connector polynucleotides.

50. The method of claim 1, wherein said at least 2 connector polynucleotides comprise connector polynucleotides comprising a sequence of n nucleotides, wherein n is an integer of from 8 to less than 100.

51. The method of claim 50, wherein said at least 2 connector polynucleotides further comprise connector polynucleotides comprising at least 1 branching point connecting at least three polynucleotide fragments comprising a sequence of n nucleotides, wherein n is an integer of from 8 to less than 100.

52. The method of claim 1, wherein said at least 2 complementary connector polynucleotides comprise polynucleotides comprising a sequence of n nucleotides, wherein n is an integer of from 8 to less than 60.

53. The method of claim 52, wherein said at least 2 complementary connector polynucleotides further comprise polynucleotides comprising at least 1 branching point connecting at least three polynucleotide fragments comprising a sequence of n nucleotides, wherein n is an integer of from 8 to less than 60.

54. The method of claim 1, wherein the polynucleotide part of at least one connector polynucleotide and/or at least one complementary connector polynucleotide is capable of undergoing self-hybridization.

55. The method of claim 1 comprising the further step of covalently linking at least one connector polynucleotide to at least one complementary connector polynucleotide.

56. The method of claim 1, wherein the connector polynucleotides and/or the complementary connector polynucleotides are provided in batch.

57. The method of claim 1, wherein the connector polynucleotides and/or the complementary connector polynucleotides are provided sequentially, and wherein at least some functional entities provided with the connector polynucleotides and/or with the complementary connector polynucleotides are reacted before additional connector polynucleotides and/or the complementary connector polynucleotides are provided.

58. The method of claim 57, wherein functional entities are reacted when a) at least two connector polynucleotides comprising at least two functional entities have been provided, and/or b) at least two complementary connector polynucleotides comprising at least two functional entities have been provided, and/or c) when at least one connector polynucleotide comprising at least one functional entity and at least one complementary connector polynucleotide comprising at least one functional entity have been provided.

59. A method for synthesising a plurality of different bifunctional molecules each comprising a molecule part linked to a nucleic acid part, the method comprising the steps of:

i) providing a plurality of connector polynucleotides each capable of hybridizing to at least 1 complementary connector polynucleotide,

ii) providing a plurality of complementary connector polynucleotides each capable of hybridizing to at least 1 connector polynucleotide,

iii) hybridizing the plurality of connector polynucleotides and complementary connector polynucleotides, thereby forming a plurality of different nucleic acid parts as hybridisation complexes, each hybridisation complex comprising at least 2 complementary connector polynucleotides and at least 2 connector polynucleotides, wherein, for each of said hybridisation complexes, at least 2 of said complementary connector polynucleotides comprise at least 1 functional entity, and at least 1 of said complementary connector polynucleotides hybridizes to at least 2 connector polynucleotides, and with the proviso that no single complementary connector or connector polynucleotide of the hybridisation complex hybridizes to all of the remaining complementary connector or connector polynucleotides of the hybridisation complex,

iv) reacting at least 2 functional entities of each hybridisation complex, wherein, for each hybridisation complex, the reaction of the functional entities results in the formation of a different molecule part comprising a different molecule by covalently linking at least 2 functional entities provided by separate complementary connector polynucleotides,

thereby providing a plurality of different bifunctional molecules wherein the nucleic acid part of each bifunctional molecule comprises nucleotide sequences that identify the functional entities that covalently reacted to form the molecule comprised within the molecule part.

60. The method of claim 59 comprising the further step of selecting molecules having desirable characteristics, wherein the selection employs a predetermined assaying procedure.

61. The method of claim 59 comprising the further step of amplifying at least part of the individual connector polynucleotides used for the synthesis of a selected molecule, wherein optionally at least one PCR primer comprises a functional entity and further optionally also part of the polynucleotide part of a connector polynucleotide.

62. The method of claim 61 comprising the further step of contacting a population of said amplified connector polynucleotides, or fragments thereof, with a plurality of complementary connector polynucleotides.

63. The method of claim 62 comprising the further step of performing an additional synthesis round using a population of said amplified connector polynucleotides or a population of said amplified connector polynucleotide fragments.

64. The method of claim 59 comprising the further steps of ligating, optionally preceded by a polynucleotide extension reaction, individual connector polynucleotides, and ligating, optionally preceded by performing a polynucleotide extension reaction, individual complementary connector polynucleotides, wherein said ligation results in linking individual connector polynucleotides and/or individual complementary connector polynucleotides by covalent bonds.

65. The method of claim 64 comprising the further steps of

a) digesting said ligated connector polynucleotides and complementary connector polynucleotides,

b) displacing the duplex polynucleotide strands generated by the ligation reaction, thereby generating single polynucleotide strands of ligated connector polynucleotides and ligated complementary connector polynucleotides, and

c) contacting the single stranded polynucleotides generated in step b) with a plurality of complementary connector polynucleotides at least some of which comprises at least one functional entity comprising a reactive group.

66. The method of claim 65 comprising the further step of performing an additional synthesis round, using as starting materials the population of connector polynucleotides obtained in step b) of claim 65, and the plurality of complementary connector polynucleotides provided in step c) of claim 65.

67. The method of claim 59, wherein the plurality of complementary connector polynucleotides comprises from about 20 to about 10⁶ different complementary connector polynucleotides.

68. The method of claim 59 comprising the further steps of linking individual connector polynucleotides by ligation and/or linking individual complementary connector polynucleotides by ligation, synthesising a plurality of different molecules by reacting for each hybridization complex reactive groups of different functional entities, wherein each of said molecules are linked to a polynucleotide of the hybridization complex, selecting and/or isolating desirable molecules linked to a polynucleotide of the hybridization complex by a predetermined selection procedure, including a binding assay, isolating from selected and/or isolated hybridization complexes polynucleotides comprising individual connector polynucleotides linked by ligation, optionally amplifying said polynucleotides, digesting said polynucleotides comprising individual connector polynucleotides and obtaining a plurality of connector polynucleotides, and contacting the plurality of connector polynucleotides generated in step e) with a plurality of complementary connector polynucleotides at least some of which comprises at least one functional entity, and performing a second or further round molecule synthesis using said plurality of connector polynucleotides and said plurality of complementary connector polynucleotides.

69. The method of claim 68, wherein steps a) and b) are performed sequentially in any order.

70. The method of claim 68, wherein steps a) and b) are performed simultaneously.

71. The method of claim 68, wherein steps a) and c) are performed sequentially in any order.

72. The method of claim 68, wherein steps a) and c) are performed simultaneously.

73. The method of claim 59 comprising the further steps of linking individual connector polynucleotides by ligation and/or linking individual complementary connector polynucleotides by ligation, synthesising a plurality of different molecules by reacting for each hybridization complex reactive groups of different functional entities, wherein each of said molecules are linked to a polynucleotide of the hybridization complex, selecting and/or isolating desirable molecules linked to a polynucleotide of the hybridization complex by a predetermined selection procedure, including a binding assay, isolating from selected and/or isolated hybridization complexes polynucleotides comprising individual connector polynucleotides linked by ligation, optionally amplifying said polynucleotides, contacting the plurality of polynucleotides comprising connector polynucleotides linked by ligation generated in step d) with a plurality of complementary connector polynucleotides each comprising at least one functional entity, performing a second or further round molecule synthesis using said plurality of connector polynucleotides and said plurality of complementary connector polynucleotides, and optionally repeating steps c) to f).

74. The method of claim 59, wherein the plurality of synthesised molecules are selected from the group consisting of α

-peptides, β

-peptides, γ

-peptides, ω

-peptides, mono-, di- and tri-substituted α

-peptides, β

-peptides, γ

-peptides, ω

-peptides wherein the amino acid residues are in the La-form or in the fl-form, vinylogous polypeptides, glycopoly-peptides, polyamides, vinylogous sulfonamide peptides, polysulfonamides, conjugated peptides comprising prosthetic groups, polyesters, polysaccharides, polycarbamates, polycarbonates, polyureas, polypeptidylphosphonates, polyurethanes, azatides, oligo N-substituted glycines, polyethers, ethoxyformacetal oligomers, poly-thioethers, polyethylene glycols (PEG), polyethylenes, polydisulfides, polyarylene sulfides, polynucleotides, PNAs, LNAs, morpholinos, oligo pyrrolinones, polyoximes, polyimines, polyethyleneimines, polyimides, polyacetals, polyacetates, polystyrenes, polyvinyl, lipids, phospholipids, glycolipids, polycyclic compounds comprising e.g. aliphatic or aromatic cycles, including polyheterocyclic compounds, proteoglycans, and polysiloxanes, including any combination thereof,

wherein each molecule is synthesised by reacting a plurality of functional entities preferably in the range of from 2 to 200,

wherein the functional entities of the above molecules can be linked by a chemical bond selected from the group of chemical bonds consisting of peptide bonds, sulfonamide bonds, ester bonds, saccharide bonds, carbamate bonds, carbonate bonds, urea bonds, phosphonate bonds, urethane bonds, azatide bonds, peptoid bonds, ether bonds, ethoxy bonds, thioether bonds, single carbon bonds, double carbon bonds, triple carbon bonds, disulfide bonds, sulfide bonds, phosphodiester bonds, oxime bonds, imine bonds, imide bonds, including any combination thereof,

or wherein the backbone structure of a synthesised molecule preferably comprises or essentially consists of one or more molecular group(s) selected from —

NHN(R)C0-;

—

NHB(R)CO—

;

—

NHC(RR′

)CO—

;

—

NHC(═

CHR)CO—

;

—

NHC₆H₄CO—

;

—

NHCH₂CHRCO—

;

—

NHCHRCH₂CO—

;

—

COCH₂—

;

—

COS—

;

—

CONR—

;

—

COO—

;

—

CSNH—

;

—

CH₂NH—

;

—

CH₂CH₂—

;

—

CH₂S—

;

—

CH₂SO—

;

—

CH₂SO₂—

;

—

CH(CH₃)S—

;

—

CH═

CH—

;

—

NHCO—

;

—

NHCONH—

;

—

CONHO—

;

—

C(═

CH₂)CH₂⁻

;

—

PO₂NH—

;

—

PO₂CH₂—

;

—

PO₂CH₂N⁺—

;

—

SO₂NH⁻

—

; and

lactams, including any combination thereof.

75. The method of claim 59, wherein said method results in the synthesis of more than or about 10³ different molecules.

76. A bifunctional molecule comprising a molecule part linked to a nucleic acid part, wherein

the molecule part comprises a molecule formed from the covalent reaction of two or more different functional entities, wherein

the nucleic acid part comprises a hybridization complex formed by the hybridization between at least two connector polynucleotides and at least two complimentary connector polynucleotides and wherein the nucleic acid part comprises nucleotide sequences that identify the different functional entities that reacted to form the molecule, wherein

at least two of the connector polynucleotides are linked to the molecule part, wherein

at least one of the connector polynucleotides linked to the molecule part hybridizes to at least two complementary connector polynucleotides, and

with the proviso that no single connector or complementary connector polynucleotide of the hybridization complex hybridizes to all of the remaining connector or complementary connector polynucleotides of the hybridization complex.

77. The bifunctional molecule according to claim 76 comprising at least n connector polynucleotides and at least n−

1 complementary connector polynucleotides, n being an integer of from 3 to 6, wherein each complementary connector polynucleotide hybridizes to at least 2 connector polynucleotides.

78. The bifunctional molecule according to claim 77, wherein n is 3 or 4.

79. The bifunctional molecule according to claim 76 comprising at least n connector polynucleotides and at least n complementary connector polynucleotides, n being an integer of from 3 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

80. The bifunctional molecule according to claim 79, wherein n complementary connector polynucleotides hybridize to at least 2 connector polynucleotides.

81. The bifunctional molecule according to claim 79, wherein n is 3 or 4.

82. The bifunctional molecule according to claim 76 comprising at least n connector polynucleotides and at least n+1 complementary connector polynucleotides, n being an integer of from 3 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

83. The bifunctional molecule according to claim 82, wherein n complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

84. The bifunctional molecule according to claim 82, wherein n is 3 or 4.

85. The bifunctional molecule according to claim 76 comprising at least n connector polynucleotides and at least n+2 complementary connector polynucleotides, n being an integer of from 3 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

86. The bifunctional molecule according to claim 85, wherein n complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

87. The bifunctional molecule according to claim 85, wherein n is 3 or 4.

88. The bifunctional molecule according to claim 76 comprising at least n connector polynucleotides and at least n+3 complementary connector polynucleotides, n being an integer of from 3 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

89. The bifunctional molecule according to claim 88, wherein n complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

90. The bifunctional molecule according to claim 88, wherein n is 3 or 4.

91. The bifunctional molecule according to claim 76 comprising at least n connector polynucleotides and at least n+4 complementary connector polynucleotides, n being an integer of from 3 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

92. The bifunctional molecule according to claim 91, wherein n complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

93. The bifunctional molecule according to claim 91, wherein n is 3 or 4.

94. The bifunctional molecule according to claim 76, wherein said at least two connector polynucleotides comprises branched connector polynucleotides, wherein at least n branched connector polynucleotides and at least n complementary connector polynucleotides are provided, n being an integer of from 2 to 6, and wherein at least n−

1 complementary connector polynucleotide hybridize to at least 2 branched connector polynucleotides.

95. The bifunctional molecule according to claim 94 comprising at least n+1 complementary connector polynucleotides.

96. The bifunctional molecule according to claim 94, wherein at least n complementary connector polynucleotides hybridize to at least 2 branched connector polynucleotides.

97. The bifunctional molecule according to claim 96, wherein at least n+1 complementary connector polynucleotide hybridize to at least 2 connector polynucleotides.

98. The bifunctional molecule according to claim 94, wherein n is 3 or 4.

99. A composition or plurality of bifunctional molecules according to claim 76.

100. The composition or plurality according to claim 99 comprising at least about 10³ different bifunctional molecules.

101. The bifunctional molecule according to claim 76,

wherein the said molecule part is selected from the group consisting of α

-peptides, β

-peptides, γ

-peptides, ω

-peptides, vinylogous polypeptides, glycopoly-peptides, polyamides, vinylogous sulfonamide peptides, polysulfonamides, conjugated peptides comprising prosthetic groups;

polyesters, polysaccharides, polycarbamates, polycarbonates, polyureas, polypeptidylphosphonates, polyurethanes, azatides, oligo N-substituted glycines, polyethers, ethoxyformacetal oligomers, poly-thioethers, polyethylene glycols (PEG), polyethylenes, polydisulfides, polyarylene sulfides, polynucleotides, PNAs, LNAs, morpholinos, oligo pyrrolinones, polyoximes, polyimines, polyethyleneimines, polyimides, polyacetals, polyacetates, polystyrenes, polyvinyl, lipids, phospholipids, glycolipids, polycyclic compounds comprising aliphatic or aromatic cycles;

proteoglycans, and polysiloxanes, and any combination thereof.

102. The bifunctional molecule according to claim 76, wherein the molecule part comprises a cyclic sequence of functional entities.

103. The bifunctional molecule according to claim 76, wherein each functional entity comprises one or more amino acid residues.

104. The bifunctional molecule according to claim 76, wherein the molecule part comprises a small molecule having a molecular weight of less than 1000 Daltons.

105. A method for selecting at least one bifunctional molecule from the composition of bifunctional molecules according to claim 99, said method comprising the steps of

a) targeting a plurality of bifunctional molecules to a potential binding partner, and

b) selecting or identifying at least one of said bifunctional molecules having an affinity for said binding partner.

106. The method of claim 105, wherein the identification of the bifunctional molecule comprises the steps of decoding the nucleic acid sequence of isolated connector polynucleotides to reveal the identity of functional entities that have participated in the formation of the molecule(s) having an affinity for said binding partner.

107. A method for evolving a plurality of bifunctional molecules according to claim 76, said method comprising the steps of

a) selecting at least one bifunctional molecule,

b) isolating connector polynucleotides, or fragments of such polynucleotides, from said bifunctional molecule,

c) providing a plurality of complementary connector polynucleotides,

d) hybridising said isolated connector polynucleotides and said plurality of complementary connector polynucleotides,

e) reacting functional entities of said complementary connector polynucleotides,

f) optionally repeating any combination of the aforementioned steps, and

g) evolving a plurality of bifunctional molecules each comprising a different molecule comprising covalently linked functional entities.

108. A method for synthesising at least one bifunctional molecule comprising a molecule part linked to a nucleic acid part, said method comprising the steps of

i) providing at least 4 building block polynucleotides each capable of hybridizing to at least 1 other building block polynucleotide and wherein at least two of said building block polynucleotides comprise at least one functional entity,

a)

b)

c)

ii) forming the nucleic acid part as a hybridization complex comprising the at least 4 building block polynucleotides, wherein at least 2 of said building block polynucleotides comprise at least 1 functional entity, wherein at least 1 of said building block polynucleotide hybridizes to at least 2 other building block polynucleotides, and with the proviso that no single building block polynucleotide of the hybridization complex hybridizes to all of the remaining building block polynucleotides of the hybridization complex,

iii) synthesising a molecule of the molecule part by covalently reacting the at least 2 functional entities;

thereby providing the at least one bifunctional molecule wherein the nucleic acid part of the bifunctional molecule comprises nucleotide sequences that identify the functional entities that covalently reacted to form the molecule of that bifunctional molecule.

109. The method of claim 108, comprising the steps of

i) providing m building block polynucleotides selected from the group consisting of

building block polynucleotides comprising at least 1 functional entity, and

building block polynucleotides comprising at least 1 spacer region and no functional entity,

wherein m is an integer of at least 4 and less than 200,

ii) hybridizing the m building block polynucleotides to form a hybridization complex,

wherein at least 2 of said building block polynucleotides comprise at least 1 functional entity,

wherein at least 1 of said building block polynucleotides hybridizes to at least 2 other building block polynucleotides,

with the proviso that no single building block polynucleotide hybridizes to the remaining m−

1 building block polynucleotides,

iii) reacting at least 3 functional entities,

wherein the reaction of said functional entities results in the formation of the molecule by covalently linking at least 2 functional entities provided by separate building block polynucleotides.

110. The method of claim 109, wherein m is 4, and wherein the complex comprises

i) p building block polynucleotides comprising at least 1 functional entity, q building block polynucleotides comprising at least 1 functional entity, and

iii) r building block polynucleotides comprising at least 1 spacer region and no functional entity,

wherein p+q+r is 4,

wherein p is an integer from 2 to 4,

wherein q is an integer from 0 to 2,

wherein the sum of p and q is 4 or less, and

wherein the value of r is given by r=4−

(p+q).

111. The method of claim 110, wherein the sum of q and r is at least 1.

112. The method of claim 109, wherein m is 6, and wherein the complex comprises

i) p building block polynucleotides comprising at least 1 functional entity,

ii) q building block polynucleotides comprising at least 1 functional entity, and

iii) r building block polynucleotides comprising at least 1 spacer region and no functional entity,

wherein p+q+r is 6,

wherein p is an integer from 2 to 6,

wherein q is an integer from 0 to 4,

wherein the sum of p and q is 6 or less, and

wherein the value of r is given by r=6−

(p+q).

113. The method of claim 109, wherein m is 8, and wherein the complex comprises

i) p building block polynucleotides comprising at least 1 functional entity,

ii) q building block polynucleotides comprising at least 1 functional entity, and

iii) r building block polynucleotides comprising at least 1 spacer region and no functional entity,

wherein p+q+r is 8,

wherein p is an integer from 3 to 8,

wherein q is an integer from 0 to 5,

wherein the sum of p and q is 8 or less, and

wherein the value of r is given by r=8−

(p+q).

114. The method of claim 109, wherein at least 3 of said building block polynucleotides comprise at least 1 functional entity,

wherein the number of building block polynucleotides hybridizing to at least 2 other building block polynucleotides is in the range of from 1 to m,

with the proviso that no single building block polynucleotide hybridises to the remaining m−

1 building block polynucleotides.

115. A method for synthesising a plurality of different bifunctional molecules each comprising a molecule part linked to a nucleic acid part, said method comprising the steps of

i) providing a plurality of at least 1000 different building block polynucleotides each comprising at least one functional entity,

ii) selecting or providing from said plurality of building block polynucleotides, n different building block polynucleotides for the synthesis of a different molecule of the molecule part, wherein n is an integer of at least 4 and less than 200,

iii) optionally further providing a plurality of building block polynucleotides selected from the group consisting of building block polynucleotides comprising at least 1 functional entity and building block polynucleotides having no functional entity,

iv) hybridizing at least the selected or provided n building block polynucleotides to form the nucleic acid part as a hybridization complex,

wherein at least n of said building block polynucleotides comprise at least 1 functional entity,

wherein at least 1 of said building block polynucleotides hybridizes to at least 2 other building block polynucleotides,

with the proviso that no single building block polynucleotide hybridizes to the remaining n−

1 building block polynucleotides, and

v) covalently reacting the at least n functional entities, wherein the covalent reaction of said functional entities provided by separate building block polynucleotides results in the formation of at least one molecule, wherein the at least one molecule is linked to at least one building block polynucleotide, and repeating the steps ii) to v) for different selections or provisions of building block polynucleotides each comprising at least one functional entity, thereby generating a plurality of different bifunctional molecules wherein the nucleic acid part of the bifunctional molecule comprises nucleotide sequences that identify the functional entities that covalently reacted to form the molecule of that bifunctional molecule.

116. The method of claim 115 comprising the further steps of targeting the plurality of bifunctional molecules obtained from the method of claim 115 to at least one binding partner for at least one of said molecule parts of said bifunctional molecules, selecting at least one bifunctional molecule having an increased affinity for said binding partner, and identifying the molecule part of the bifunctional molecule by decoding the polynucleotide part of the plurality of building block polynucleotides forming the hybridisation complex of said bifunctional molecule.

117. The method of claim 116 comprising the further step of improving the binding of said molecule part to said binding partner, said improvement comprising the steps of isolating building block polynucleotides from the isolated bifunctional molecule, optionally separating building block polynucleotides into fractions depending on whether or not they have donated a reactant to the synthesis of the at least one molecule, hybridising some or all of said isolated building block polynucleotides with a plurality of building block polynucleotides each comprising at least one reactant, forming a plurality of second or further bifunctional molecules by reacting said reactants and linking said molecules to at least one building block polynucleotide of their respective hybridisation complexes, targeting said plurality of second or further bifunctional molecules to at least one target comprising a conceivable binding partner for the molecule parts of said plurality of bifunctional molecules, and selecting bifunctional molecules having improved binding affinities for said at least one target.

118. A bifunctional molecule comprising a molecule part linked to a nucleic acid part, wherein

the molecule part comprises a molecule formed by a covalent reaction of different functional entities, and wherein

the nucleic acid part comprises a hybridization complex of at least four building block polynucleotides, each building block polynucleotide comprising a nucleotide sequence that identifies a functional entity that reacted to form the molecule, wherein

at least two of the building block polynucleotides hybridize to at least two of the other building block polynucleotides, wherein

the building block polynucleotides are not linked by covalent bonds when the functional entities have reacted to form the molecule of the molecule part, and wherein

the reacted functional entities are not all linked by, or do not all form, phosphodiester bonds,

with the proviso that no single building block polynucleotide of the hybridization complex hybridizes to all of the remaining building block polynucleotides of the hybridization complex.

119. The bifunctional molecule according to claim 118, wherein the molecule part comprises a cyclic sequence of functional entities.

120. The bifunctional molecule according to claim 118, wherein each functional entity comprises one or more amino acid residues.

121. The bifunctional molecule according to claim 118, wherein the molecule part comprises a small molecule having a molecular weight of less than 1000 Daltons.

122. A bifunctional molecule comprising a single molecule part linked to a nucleic acid part, wherein

the molecule part comprises a molecule formed by a covalent reaction of different functional entities, and wherein

the nucleic acid part comprises a hybridization complex of at least two connector polynucleotides hybridized to at least two complementary connector polynucleotides and wherein the nucleic acid part comprises nucleotide sequences that identify the different functional entities that reacted to form the molecule, wherein

at least one connector polynucleotide hybridizes to two complementary connector polynucleotides and wherein at least one complementary connector polynucleotide hybridizes to two connector polynucleotides, wherein

said connector polynucleotides are not linked by covalent bonds when the functional entities have reacted to form the molecule of the molecule part, wherein

the molecule part is covalently linked to at least one complimentary connector polynucleotide of the nucleic acid part, and wherein

the reacted functional entities are not all linked by, or do not all form, phosphodiester bonds,

with the proviso that no single connector or complimentary connector polynucleotide of the hybridization complex hybridizes to all of the remaining connector or complimentary connector polynucleotides of the hybridization complex.

123. A bifunctional molecule comprising a molecule part linked to a nucleic acid part, wherein

the molecule part comprises a molecule formed by a covalent reaction of different functional entities, and wherein

the nucleic acid part comprises a hybridization complex of at least two connector polynucleotides hybridized to at least two complementary connector polynucleotides and wherein the nucleic acid part comprises nucleotide sequences that identify the different functional entities that reacted to form the molecule, wherein

at least one connector polynucleotide hybridizes to two complementary connector polynucleotides and wherein at least one complementary connector polynucleotide hybridizes to two connector polynucleotides, wherein

said complementary connector polynucleotides are not linked by covalent bonds when the functional entities have reacted to form the molecule of the molecule part, wherein

the molecule part is covalently linked to at least one connector polynucleotide or complimentary connector polynucleotide of the nucleic acid part, and wherein

the reacted functional entities are not all linked by, or do not all form, phosphodiester bonds,

with the proviso that no single connector or complimentary connector polynucleotide of the hybridization complex hybridizes to all of the remaining connector or complimentary connector polynucleotides of the hybridization complex.

124. The bifunctional molecule according to claim 123, wherein the molecule part comprises a cyclic sequence of functional entities.

125. The bifunctional molecule according to claim 123, wherein the functional entities comprise amino acid residues.

126. The bifunctional molecule according to claim 123, wherein the molecule part comprises a small molecule having a molecular weight of less than 1000 Daltons.

127. A bifunctional molecule comprising a molecule part linked to a nucleic acid part, wherein

the molecule part comprises a molecule formed by a covalent reaction of different functional entities, and wherein

the nucleic acid part comprises a hybridization complex of at least two connector polynucleotides hybridized to at least two complementary connector polynucleotides and wherein the nucleic acid part comprises nucleotide sequences that identify the different functional entities that reacted to form the molecule, wherein

at least one connector polynucleotide hybridizes to two complementary connector polynucleotides and wherein at least one complementary connector polynucleotide hybridizes to two connector polynucleotides, wherein

the molecule part is covalently linked to at least one connector polynucleotide or complimentary connector polynucleotide of the nucleic acid part, wherein

said connector polynucleotides and said complementary connector polynucleotides are not linked by covalent bonds when the functional entities have reacted to form the molecule of the molecule part, and wherein

the reacted functional entities are not all linked by, or do not all form, phosphodiester bonds,

with the proviso that no single connector or complimentary connector polynucleotide of the hybridization complex hybridizes to all of the remaining connector or complimentary connector polynucleotides of the hybridization complex.

128. The bifunctional molecule according to claim 127, wherein the molecule part comprises a cyclic sequence of functional entities.

129. The bifunctional molecule according to claim 127, wherein the functional entities comprise amino acid residues.

130. The bifunctional molecule according to claim 127, wherein the molecule part comprises a small molecule having a molecular weight of less than 1000 Daltons.

131. A bifunctional molecule comprising a molecule part linked to a nucleic acid part, wherein

the molecule part comprises a molecule formed by a covalent reaction of different functional entities,

wherein the nucleic acid part comprises a hybridization complex of at least four building block polynucleotides, and wherein the nucleic acid part comprises nucleotide sequences that identify the different functional entities that reacted to form the molecule,

wherein at least two of said building block polynucleotides comprise at least one functional entity, the reaction of the reactive groups resulting in the formation of the molecule part;

wherein at least one of the building block polynucleotides comprising the at least one functional entity hybridizes to at least two other building block polynucleotides, and with the proviso that no single building block polynucleotide of the hybridization complex hybridizes to all of the remaining building block polynucleotides.

132. The bifunctional molecule according to claim 131, wherein at least 3 of said building block polynucleotides comprise at least one functional entity, the reaction of the functional entities resulting in the formation of the molecule part.

133. The bifunctional molecule according to claim 131, wherein at least 4 of said building block polynucleotides comprise at least one functional entity, the reaction of the functional entities resulting in the formation of the molecule part.