Method for the synthesis of a bifunctional complex

• US 8,206,901 B2
• Filed: 10/30/2003
• Issued: 06/26/2012
• Est. Priority Date: 10/30/2002
• Status: Active Grant

• Current Assignee: Nuevolution A/S (Amgen Inc.)
• Original Assignee: Nuevolution A/S (Amgen Inc.)
• Inventors: Freskgard, Per-Ola, Franch, Thomas, Gouliaev, Alex Haahr, Lundorf, Mikkel Dybro, Felding, Jakob, Olsen, Eva Kampmann, Holtmann, Anette, Jakobsen, Soeren Nyboe, Sams, Christian Klarner, Glad, Sanne Schroeder, Jensen, Kim Birkebaek, Pedersen, Henrik
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US10/525,817
• Publication Number:
  US 20060099592A1
• Time in Patent Office: 3,162 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 obtaining a bifunctional complex comprising a display molecule part and a coding part, wherein a nascent bifunctional complex comprising a chemical reaction site and a priming site for enzymatic addition of a tag is reacted at the chemical reaction site with one or more reactants, and provided with respective tag(s) identifying the reactants(s) at the priming site using one or more enzymes.

Claims

1. A method for obtaining a library comprising bifunctional complexes, wherein each complex comprises a display molecule part and a coding part, said method comprising the steps of:

a) reacting a nascent bifunctional complex comprising a chemical reaction site and a priming site at the chemical reaction site with one or more reactants; and

(b) reacting the priming site with one or more tags identifying the one or more reactants, the reaction being catalyzed by an enzyme;

wherein steps (a) and (b) provide an intermediate bifunctional complex having a modified chemical reaction site containing a structural entity formed from the nascent bifunctional complex and the one or more reactants and a modified priming site containing the one or more tags;

(c) reacting the intermediate bifunctional complex with one or more additional reactants at the modified reactant site and one or more additional tags identifying the one or more additional reactants at the modified priming site using the methods of steps (a) and (b);

(d) optionally repeating step (c) as many times as desired; and

(e) obtaining a library which contains from 10⁴ to 10²⁰ different bifunctional complexes;

wherein the library is produced using split and mix methods, and

wherein the coding part comprises an identifier oligonucleotide comprising tags identifying the reactants which have participated in the formation of the display molecule, and a reactant and the tag identifying the reactant are not linked prior to their reaction with the chemical reaction site and the priming site, respectively, of the nascent bifunctional complex.

2. The method of claim 1, wherein the bifunctional complexes comprise a reaction product and an identifier oligonucleotide comprising tags identifying the reactants which have participated in the formation of the reaction product.

3. The method of claim 1, wherein the sequence of the tags of the identifier oligonucleotide of the bifunctional complex is used to decipher the structure of the reactants that have participated in the formation of the display molecule.

4. The method of claim 1, wherein the order of the tags determines the order of incorporation of reactants into the display molecule.

5. The method of claim 1, wherein a single reactant is reacted with the chemical reaction site in a reaction cycle, said reaction cycle further comprising adding a single tag identifying the reactant with the priming site wherein one or more enzymes catalyzes the addition of the tag.

6. The method of claim 1, wherein multiple reactants are reacted with the chemical reaction site in a reaction cycle, said reaction cycle further comprising adding multiple respective tags identifying the reactants with the priming site wherein one or more enzymes catalyzes the addition of the tags.

7. The method of claim 1, wherein the bifunctional complex is prepared by sequentially adding a tag and reacting a reactant.

8. The method of claim 1, wherein the chemical reaction site comprises a single reactive group.

9. The method of claim 1, wherein the chemical reaction site comprises a scaffold having one or more reactive groups attached.

10. The method of claim 9, wherein there is provided a scaffold “

X”

linked to a tag “

x”

identifying said scaffold, wherein a reactant “

A”

is reacted with scaffold “

X”

, said reaction resulting in “

A”

being linked to “

X”

, and wherein a tag “

a”

is added to the scaffold tag “

x”

.

11. The method of claim 9, wherein the scaffold is selected from the group consisting of benzodiazepines, steroids, hydantoins, piperazines, diketopiperazines, morpholines, tropanes, cumarines, quinolines, indoles, furans, pyrroles, oxazoles, amino acid precursors, and thiazoles.

12. The method of claim 1, wherein the chemical reaction site comprises multiple reactive groups capable of reacting with one or more reactants.

13. The method of claim 12, wherein the at least one of the multiple reactive groups is selected from the group consisting of hydroxyl groups, carboxylic acid groups, aldehyde groups, thiol groups, isocyanate groups, amine groups, ester groups, thioester groups, alkylating agents and acylating agents.

14. The method of claim 1, wherein a reactant among the one or more reactants comprises a reactive group or an activatable reactive group precursor.

15. The method of claim 1, wherein the reaction of a reactant and the chemical reaction site takes place in the presence of a further reactant mediating a connection between the reactant and the chemical reaction site.

16. The method of claim 1, wherein the reactant among the one or more reactants is a precursor for a structural entity incorporated into the display molecule.

17. The method of claim 1, wherein the number of reactive groups of the one or more reactants is from one to ten.

18. The method of claim 17, wherein the one or more reactive groups include an amino group.

19. The method of claim 17, wherein the one or more reactive groups include a carboxylic acid.

20. The method of claim 17, wherein the one or more reactive groups include a thio group.

21. The method of claim 17, wherein the one or more reactive groups include an aldehyde.

22. The method of claim 17, wherein the one or more reactive groups include a hydroxyl groups.

23. The method of claim 1, wherein the display molecule comprises a polymer or a scaffold, and a reactant featuring only one reactive group is used in the end positions of the polymers or the scaffolds.

24. The method of claim 1, wherein the display molecule comprises a polymer or a scaffold, and a reactant having two reactive groups is used for the formation of the body part of a polymer or scaffolds capable of being reacted further.

25. The method of claim 1, wherein two or more reactive groups are present on a scaffold in the form of a core structure, and different molecules of the scaffold are reacted with different reactants thereby generating different, scaffolded display molecules.

26. The method of claim 25, wherein reactants reacting with the scaffold contain one, two or several reactive groups capable of forming a connection with the scaffold.

27. The method of claim 1, wherein the nascent bifunctional complex further comprises a linking moiety, connecting the chemical reaction site and the priming site.

28. The method of claim 27, wherein the linking moiety of the nascent bifunctional complex comprises information relating to the identity of the chemical reaction site.

29. The method of claim 27, wherein the linking moiety is a nucleic acid sequence to which a complementing oligonucleotide can hybridise.

30. The method of claim 27, wherein the linking moiety is attached to the chemical reaction site via a spacer comprising a selectively cleavable linker.

31. The method of claim 30, wherein the selectively cleavable linker is cleaved and the display molecule is detached from the identifier oligonucleotide of the bifunctional complex after the formation of a final bifunctional complex.

32. The method of claim 30, wherein the selectively cleavable linker is cleaved by electromagnetic radiation.

33. The method of claim 31, wherein the bifunctional complex comprises a selectively cleavable linker capable of being cleaved by electromagnetic radiation, wherein said bifunctional complex is selected from the group consisting of

34. The method of claim 1, wherein the bifunctional complex comprises a selectively cleavable linker capable of being cleaved by UV radiation, wherein said bifunctional complex has the formula

35. The method of claim 34, wherein R¹ is the display molecule and R² is the identifier oligonucleotide comprising tags identifying the reactants which have participated in the formation of the display molecule.

36. The method of claim 1, wherein the bifunctional complex comprises a selectively cleavable linker capable of being cleaved by chemical agents, wherein said bifunctional complex has the formula

37. The method of claim 30, wherein the selectively cleavable linker is a disulfide linker.

38. The method of claim 30, wherein the selectively cleavable linker is cleaved by a protease.

39. The method of claim 1, wherein the identifier oligonucleotide comprises 3, 4, 5, or more tags.

40. The method of claim 1, wherein the priming site comprises one, two, or more nucleotides, to which a complementing oligonucleotide can hybridise.

41. The method of claim 40, wherein the priming site comprises a 3′

-OH or 5′

-phosphate group of a nucleotide.

42. The method of claim 1, wherein all tags of the identifier oligonucleotide are attached using an enzymatically catalysed reaction.

43. The method of claim 1, wherein the tags are unique tags.

44. The method of claim 1, wherein the identifier oligonucleotide of the bifunctional complex comprises a double stranded oligonucleotide in order to reduce reaction between the oligonucleotide and the reactants.

45. The method of claim 1, wherein the identifier oligonucleotide of the bifunctional complex is amplifiable.

46. The method of claim 1, wherein the nucleotides of the identifier oligonucleotide are selected from DNA and RNA.

47. The method of claim 1, wherein the nucleotides of the identifier oligonucleotide are composed of a nucleobase moiety and a backbone unit, wherein the backbone unit is composed of a sugar moiety and an internucleoside linker.

48. The method of claim 47, wherein at least one nucleobase moiety is a naturally occurring nucleobase and at least one other nucleobase moiety is a non-naturally occurring nucleobase moiety.

49. The method of claim 47, wherein the nucleobase is selected from the group consisting of purine heterocycles, pyrimidine heterocycles and heterocyclic analogues and tautomers thereof.

50. The method of claim 48, wherein the nucleobase 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, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine and inosine.

51. The method of claim 49, wherein the nucleobase is selected from the group consisting of adenine, guanine, thymine, cytosine, 5-methylcytosine and uracil.

52. The method of claim 47, wherein the backbone units of the identifier oligonucleotide are selected independently from the group consisting of

53. The method of claim 47, wherein the sugar moiety of the backbone unit is a pentose.

54. The method of claim 53, wherein the pentose is selected from the group consisting of ribose, 2′

-deoxyribose, 2′

-O-methyl-ribose, 2′

-fluor-ribose, and 2′

-4′

-O-methylene-ribose (LNA).

55. The method of claim 53 or 54, wherein the nucleobase of the nucleotide is attached to the l′

position of the pentose.

56. The method of claim 47, wherein the internucleoside linker linking two neighbouring nucleotides of the identifier oligonucleotide is selected from the group consisting of phosphodiester bonds, phosphorothioate bonds, methylphosphonate bonds, phosphoramidate bonds, phosphotriester bonds and phosphodithioate bonds.

57. The method of claim 47, wherein the identifier oligonucleotide comprises naturally occurring nucleosides of the DNA and RNA family connected through phosphodiester linkages.

58. The method of claim 47, wherein the deoxynucleosides are selected from the group consisting of deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine, and wherein the nucleosides are selected from adenosine, guanosine, uridine, cytidine, and inosine.

59. The method of claim 1, wherein each tag codes for different reactants and wherein the structure of the display molecule is deduced by taking into account different attachment chemistries, steric hindrance and deprotection of orthogonal protection groups.

60. The method of claim 1, wherein the same tag is used for a group of reactants sharing a common property selected from the group consisting of lipophilic nature, molecular weight and attachment chemistry.

61. The method of claim 1, wherein several different tags are used for the same reactant.

62. The method of claim 1, wherein two or more tags identifying the same reactant further carry information of different reaction conditions employed for the reaction of said reactant.

63. The method of claim 1, wherein a single tag specifies two or more reactants.

64. The method of claim 1, wherein individual tags are distinguished from each other by only a single nucleotide.

65. The method of claim 1, wherein two or more differences distinguish a tag from any other tag.

66. The method of claim 1, wherein the length of the tags is 5 nucleotides, and wherein more than 100 nucleotide combinations exist for generating two or more differences between any two tags.

67. The method of claim 1, wherein the tags have from 2 to 100 nucleotides.

68. The method of claim 67, wherein the tags have from 3 to 50 nucleotides.

69. The method of claim 67, wherein the tags have from 4 to 20 nucleotides.

70. The method of claim 67, wherein the tags have from 5 to 15 nucleotides.

71. The method of claim 1, wherein each tag is separated by a binding region of from 1 to 20 nucleotides.

72. The method of claim 71, wherein each binding region identifies the position of a tag in the identifier oligonucleotide.

73. The method of claim 71, wherein the binding regions comprise one or more nucleobases forming three hydrogen bonds to a cognate nucleobase.

74. The method of claim 73, wherein said nucleobases are guanine and cytosine.

75. The method of claim 72, wherein the binding region has a backbone modification selected from the group consisting of 2′

-O-methyl substitution of a ribose moiety, 2′

-4′

-O-methylene cyclisation of the ribose moiety (Locked Nucleic Acid;

LNA), and peptide nucleic acids (PNA).

76. The method of claim 1, wherein two or more reactants are reacted with the chemical reactive site, and wherein the tags of the identifier oligonucleotide are separated by a binding region.

77. The method of claim 76, wherein the binding region is a substrate recognised by an enzyme selected from a polymerase and a ligase.

78. The method of claim 1, wherein each tag comprises nucleotides identifying a reactant and a framing sequence identifying the synthesis history of the reactant.

79. The method of claim 1, wherein the tags further comprise a flanking region comprising a signal group allowing for detection of the bifunctional complex.

80. The method of claim 79, wherein the flanking regions comprise a detectable label.

81. The method of claim 79, wherein the flanking regions comprise priming sites for PCR amplification.

82. The method of claim 1, wherein the display molecule is the reaction product of two or more reactants with the chemical reaction site.

83. The method of claim 1, wherein the display molecule is the reaction product of more than one reactant and the chemical reaction site of the bifunctional complex.

84. The method of claim 1, wherein the display molecule is a non-polymeric molecule having a molecular weight of less than 1000 Da.

85. The method of claim 1, wherein the display molecule is a non-polymeric molecule having a molecular weight of more than 1000 Da.

86. The method of claim 1, wherein the display molecule is a polymeric molecule.

87. The method of claim 1, wherein a first reactant forms an intermediate product upon reaction with the chemical reactive site and a second reactant reacts with the intermediate product to obtain the display molecule, or a precursor thereof.

88. The method of claim 1, wherein two or more reactants react with each other to form an intermediate product and the chemical reactive site reacts with this intermediate product to obtain the display molecule, or a precursor thereof.

89. The method of claim 1, wherein the enzyme used for enzymatic addition of a tag to the priming site is selected from the group consisting of a polymerase, a ligase, and a recombinase.

90. The method of claim 1, wherein the tag is attached to the priming site of a nascent bifunctional complex by using an enzymatic extension reaction.

91. The method of claim 90, wherein the extension reaction is performed by a polymerase or a ligase, or a combination thereof.

92. The method of claim 1, wherein the identifier oligonucleotide comprising tags identifying the reactants is transformed to a double stranded identifier oligonucleotide by an extension process in which a primer is annealed to the 3′

end of the identifier oligonucleotide and extended using a polymerase.

93. The method of claim 92, wherein the extension reaction uses an anti-tag oligonucleotide as primer.

94. The method of claim 92, wherein the substrate used by the polymerase is a mixture of triphosphate nucleotides selected from the group consisting of dATP, dGTP, dTTP, dCTP, rATP, rGTP, rTTP, rCTP and rUTP.

95. The method of claim 1, comprising the further step of annealing a single stranded overhang of an anti-tag oligonucleotide identifying a reactant to the 3′

end of the identifier oligonucleotide of the nascent bifunctional complex.

96. The method of claim 95, wherein the anti-tag is used as a primer for transcribing the identifier oligonucleotide of the nascent bifunctional complex to generate a double stranded oligonucleotide identifier, wherein said transcription uses a polymerase and a mixture of dNTPs.

97. The method of claim 92 or 96, wherein the polymerase is selected from the group consisting of DNA polymerase, RNA polymerase, Reverse Transcriptase, Taq DNA polymerase, Pfu polymerase, Thermococcus literalis hermostable polymerase, HIV-1 Reverse Transcriptase, and Klenow fragment.

98. The method of claim 92 or 96, wherein the polymerase is selected from polymerases allowing mismatch extension.

99. The method of claim 98, wherein the polymerases are selected from the group consisting of DNA polymerase η

and DNA polymerase τ

.

100. The method of claim 1, wherein the enzyme is a ligase.

101. The method of claim 100, wherein the substrate for the ligase is an oligonucleotide comprising two or more nucleotides.

102. The method of claim 100, wherein the ligase mediates a single stranded ligation, wherein an oligonucleotide tag is ligated to the nascent bifunctional molecule.

103. The method of claim 102, wherein the single stranded ligation is performed by ligating a 3′

-OH group of a first nucleic acid selected from the priming site and the oligonucleotide tag to a 5′

phosphate group of a second nucleic acid selected from the priming site and the oligonucleotide tag, with the proviso that the two nucleic acids carry different reactive groups capable of being ligated.

104. The method of claim 100, wherein the ligase mediates a double stranded ligation, wherein the priming site of the nascent bifunctional complex hybridises to a complementing oligonucleotide prior to ligation to an oligonucleotide tag.

105. The method of claim 104, wherein double stranded ligation takes place in the presence of a third oligonucleotide complementing part of the 3′

end and part of the 5′

end, respectively, of a first and second nucleic acid, said first and second nucleic acids being selected from the priming site and the oligonucleotide tag.

106. The method of claim 100, wherein the ligase is selected from the group consisting of Taq DNA ligase, T4 DNA ligase, T4 RNA ligase, T7 DNA ligase, and E. coli DNA ligase.

107. The method of claim 104 or 105, wherein the double stranded oligonucleotide to be ligated has blunt ends, and wherein a T4 RNA ligase is used for ligating said blunt ends.

108. The method of claim 104 or 105, wherein the double stranded oligonucleotide to be ligated has sticky ends, and wherein a Taq DNA ligase is used for ligating said sticky ends.

109. The method of claim 100, wherein a combination of polymerase transcription and ligational coupling of hybridised and complementary oligonucleotides is used for generating a double stranded identifier oligonucleotide.

110. The method of claim 109, wherein a gap in an otherwise double stranded identifier oligonucleotide is initially filled-in by a polymerase and then ligated by a ligase to an upstream oligonucleotide to produce an exclusively double stranded identifier oligonucleotide.

111. The methods of claim 1, wherein the enzyme reactions are conducted in aqueous solvent, and wherein at least some reactions between a reactant and the chemical reaction site are carried out in an organic solvent.

112. The method of claim 111, wherein the enzyme reaction is initially carried out in an aqueous solvent, the reaction mixture is then lyophilized and subsequently the reaction product is dissolved or dispersed in a different solvent prior to the reaction at the chemical reaction site.

113. The method of claim 111, wherein no lyophilization step is included in the method, and wherein appropriate reaction conditions are obtained by adding a solvent to the aqueous solvent.

114. The method of claim 113, wherein the added solvent is miscible with the aqueous solvent, wherein the addition of the solvent produces a homogeneous reaction solvent.

115. The method of claim 113, wherein the added solvent is immiscible with the aqueous solvent, wherein the addition of the solvent produces a bi-phasic reaction solvent.

116. The method of claim 1, wherein no protection groups are used to protect the identifier oligonucleotide or the display molecule of the nascent bifunctional complex.

117. The method of claim 1, wherein the display molecule of a nascent bifunctional complex is protected by protection groups.

118. The method of claim 1, wherein the reactive groups of the chemical reaction site are initially in an activatable pro-form, wherein the activatable pro-form reactive groups are activated prior to reaction with a reactant.

119. The method of claim 118, wherein the activatable pro-form reactive groups are protected with a group preventing reaction with a reactant.

120. The method of claim 1, wherein the nascent bifunctional complexes are provided in separate reaction compartments and wherein the reaction between the chemical reaction site and one or more reactants and reaction of the priming site with the one or more tags are performed in the separate reaction compartments.

121. The method of claim 120, wherein the nascent bifunctional complexes provided in each separate reaction compartment are identical.

122. The method of claim 120, wherein the nascent bifunctional complexes provided in each separate reaction compartment are different.

123. The method of claim 120, wherein the nascent bifunctional complexes differ at the chemical reaction site and wherein each of the nascent bifunctional complexes comprises a tag identifying the structure of the chemical reaction site.

124. The method of claim 120, wherein the reactants applied in each separate reaction compartment are identical.

125. The method of claim 120, wherein the reactants applied in each separate reaction compartment are different.

126. The method of claim 120, wherein the reaction conditions in each separate reaction compartment are the same.

127. The method of claim 120, wherein the reaction conditions in each separate reaction compartment are different.

128. The method of claim 120, wherein the nascent bifunctional complex is reacted with more than a single reactant.

129. The method of claim 120, wherein two or more reaction compartments are pooled together after the formation in each reaction compartment of a bifunctional complex and subsequently split into an array of further reaction compartments for a subsequent round of reaction.

130. The method of claim 129 comprising more than one round of bifunctional complex synthesis, wherein the reaction product of a preceding synthesis round is used as a nascent bifunctional complex in a subsequent synthesis round for obtaining a library of different bifunctional complexes each comprising a display molecule and an identifier oligonucleotide comprising tags identifying the reactants which have participated in the formation of individual display molecules.

131. The method of claim 130, wherein the separate reaction compartments are mixed together and split into further reaction compartments between each round of synthesis.

132. The method of claim 129, wherein two or more reactant reactions occur before a tag is added to the priming site.

133. The method of claim 130, wherein the last round of synthesis of the bifunctional complex includes the incorporation of an oligonucleotide comprising a priming site.

134. The method of claim 120, wherein the library contains from 10⁵ to 10⁸ different bifunctional complexes.

135. The method of claim 120, wherein the library contains from 10⁵ to 10¹⁰ different bifunctional complexes.

136. The method according to claim 27, wherein the linking moiety is selected from a nucleic acid sequence and polyethylene glycol (PEG).

137. The method according to claim 136, wherein the number of nucleocides in the linking moiety is 8 or more.

138. The method according to claim 9, wherein the one or more reactive groups are selected from the group consisting of amine groups, carboxylic acid groups, thio groups, aldehyde groups, and hydroxyl groups.

139. The method of claim 12, wherein the chemical reaction site undergoes an alkylation reaction.

140. The method of claim 12, wherein the chemical reaction site undergoes an acylation reaction.

141. The method of claim 12, wherein an alkylation product is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

142. The method of claim 12, wherein an arylation product is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

143. The method of claim 12, wherein an acylation product is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

144. The method of claim 12, wherein a cycloaddition product is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants, whereby a cyclic product is formed.

145. The method of claim 12, wherein an amide bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

146. The method of claim 12, wherein an carboxylic ester bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

147. The method of claim 12 wherein a sulfonamide bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

148. The method of claim 12, wherein an disulfide bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

149. The method of claim 12, wherein an S-alkyl bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

150. The method of claim 12, wherein an N-alkyl bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

151. The method of claim 12, wherein an O-alkyl bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

152. The method of claim 12, wherein an aryl-vinyl bond or an alkyne-vinyl bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

153. The method of claim 12, wherein an oxime bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

154. The method of claim 12, wherein an imine bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

155. The method of claim 1, wherein a bicyclic product is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

156. The method of claim 12, wherein a triazole product is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

157. The method of claim 12, wherein a peptide or polypeptide product is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

158. The method of claim 12, wherein a s-alkyl bond is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

159. The method of claim 12, wherein an ether product is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

160. The method of claim 12, wherein an alkyne product is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

161. The method of claim 12, wherein a vinyl product is formed by the reaction between a) the nascent bifunctional complex comprising one or more chemical reaction sites and b) one or more reactants.

162. The method claim 1, wherein the enzyme reactions are conducted in aqueous solvent, and wherein at least some reactions between a) a reactant and the chemical reaction site are carried out in water.

163. The method of claim 1, wherein a covalently linked bifunctional complex is formed.

164. The method of claim 1, wherein a branched product is formed.

165. The method of claim 1, wherein a linear product is formed.

166. The method of claim 120 comprising the further step of identifying, from the library of different bifunctional complexes, one or more display molecule(s) having a predetermined property, wherein the method for identifying the one or more display molecule(s) comprises the steps of

a) subjecting the library of bifunctional complexes produced by the method of claim 168 to a selection condition, wherein one or more display molecules having said predetermined property is partitioned from the remainder of the bifunctional complexes of the library, and

b) identifying the one or more display molecules having said predetermined property by decoding the identifier oligonucleotide of the partitioned one or more bifunctional complexes.

167. The method of claim 166, wherein the partitioned display molecules are partitioned by a method involving one or more of the steps of plastic binding, nitrocellulose filter binding, column chromatography, filtration, affinity chromatography and centrifugation.

168. The method of claim 166, wherein the identifier oligonucleotide of the bifunctional complex is in single stranded form.

169. The method of claim 166, wherein the identifier oligonucleotide of the bifunctional complex is in double stranded form.

170. The method of claim 166, wherein the selection condition involves the step of contacting the library of bifunctional complexes with a target selected from the group consisting of a protein, a peptide, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, an antigen, an antibody, a virus, a substrate, a metabolite, a transition state analog, a cofactor, an inhibitor, a drug, a dye, a nutrient, a growth factor, a cell, a tissue, an angiotensin converting enzyme, a renin, a cyclooxygenase, a 5-lipoxygenase, an IL-10 converting enzyme, a cytokine receptor, a PDGF receptor, a type II inosine monophosphate dehydrogenase, a β

-lactamase, a fungal cytochrome P-450, a bradykinin, a neutrophil elastase, a HIV protein, a nucleocapsid, VEGF, bFGF, TGFβ

, KGF, PDGF, thrombin, theophylline, caffeine, substance P, IgE, sPLA2, red blood cells, glioblastomas, fibrin clots, PBMCs, hCG, lectins, selectins, cytokines, ICP4, and complement proteins.

171. The method of claim 166, wherein the identifier oligonucleotide of the partitioned bifunctional complexes is cleaved from the display molecule after the removal of the non-binding bifunctional complexes.

172. The method of claim 171, wherein the identifier oligonucleotide of the partitioned bifunctional complexes is recovered and decoded for identifying the respective display molecules.

173. The method of claim 166, wherein a single round of selection against a specific target is followed by amplification of the partitioned bifunctional complexes variants.

174. The method of claim 173, wherein the selected bifunctional complex variants are separately tested in an assay.

175. The method of claim 166, wherein more selection rounds against a specific target are followed by amplification of the partitioned bifunctional complexes variants.

176. The method of claim 175, wherein several selection rounds employ increased stringency conditions.

177. The method of claim 173, wherein an amplification of the selected complex is performed in between each selection step.

178. The method of any of claims 173 and 177, wherein the tags of the identifier oligonucleotide are amplified using PCR and primers generating two unique cut-sites.

179. The method of claim 178, wherein the cut-sites are used for multimerization of the tags by cloning the tags of the identifier oligonucleotide into a suitable vector for sequencing.

180. The method of claim 178, wherein the PCR product resulting from the amplification is cloned directly into a suitable vector using TA cloning.

181. The method of claim 178, wherein the PCR product resulting from the amplification is introduced to a microarray in order to identify the display molecule.

182. The method of claim 120 comprising the further step of identifying, from the library of different bifunctional complexes, one or more display molecule(s) having a predetermined property, wherein the method for identifying the one or more display molecule(s) comprises the steps of

a. partitioning the library of different bifunctional complexes, and

b. identifying reaction product(s) having the predetermined property by decoding the oligonucleotide tags of the bifunctional complex.

184. The method of claim 120, wherein a plurality of nascent bifunctional complexes each comprising a chemical reaction site and a priming site capable of receiving a tag is provided,

wherein the chemical reaction site of one or more bifunctional complexes is reacted with one or more reactants,

wherein respective tag(s) identifying the reactant(s) are reacted with the priming site of the bifunctional complexes, wherein one or more enzymes catalyzes the reaction,

wherein the library of different bifunctional complexes is subjected to a partitioning condition,

wherein display molecules having a predetermined property are partitioned from the remainder of the library,

wherein display molecules having said predetermined property are selected,

wherein display molecules having said predetermined property are identified by decoding the identifier oligonucleotide of each of the bifunctional complexes.

185. The method of claim 1 wherein at least one enzymatic addition of a tag is performed using an enzyme with ligase activity.

186. The method of claim 1 wherein at least one enzymatic addition of a tag is performed using a DNA ligase.

187. The method of claim 1 wherein at least one enzymatic addition of a tag is performed using a recombinase.

188. The method of claim 1 wherein at least one enzymatic addition of a tag is performed in aqueous solution.

189. The method of claim 1 wherein at least one enzymatic addition of a tag is performed in a mixture of an aqueous solvent and a non-aqueous solvent.

190. The method of claim 1 wherein at least one reaction with a reactant is performed with the nascent bifunctional complex immobilized on a solid support and at least one other reaction with a reactant is performed with the nascent bifunctional complex not so immobilized.

191. The method of claim 1 wherein the library comprises from 10⁵ to 10²⁰ different bifunctional complexes.

192. The method of claim 1, wherein the bifunctional complex is prepared by simultaneously adding a tag and reacting a reactant.

193. The method of claim 170, wherein the HIV protein is selected from the group consisting of tat, rev, gag, int, and RT.

183. A method for obtaining a library of different bifunctional complexes each comprising a display molecule part and an identifier oligonucleotide part, said method comprising the steps of:

i) providing a nascent bifunctional complex comprising a chemical reaction site capable of receiving one or more reactants and a priming site capable of receiving one or more tags,

ii) reacting the chemical reaction site with one or more reactants, and

iii) reacting the priming site with respective tag(s) identifying the reactant(s), wherein one or more enzymes catalyze the reaction;

wherein steps (ii) and (iii) provide an intermediate bifunctional complex having a modified chemical reaction site containing a structural entity formed from the nascent bifunctional complex and the one or more reactants and a modified priming site containing the one or more tags;

iv) reacting the intermediate bifunctional complex with one or more additional reactants at the modified reactant site and one or more additional tags identifying the one or more additional reactants at the modified priming site using the methods of steps (ii) and (iii);

v) optionally repeating step (iv) as many times as desired; and

vi) obtaining a library which contains from 10⁴ to 10²⁰ different bifunctional complexes;

vii) subjecting the library of different bifunctional complexes to a partitioning condition,

viii) partitioning a display molecule, or a subset of display molecules, having a predetermined property from the remainder of the library,

ix) selecting a display molecule, or a subset of display molecules, having said predetermined property,

x) identifying display molecule(s) having said predetermined property by decoding the identifier oligonucleotide of the bifunctional complex,

xi) reacting the partitioned bifunctional complex(es) one or more times with further reactant(s) and reacting said bifunctional complexes with respective identifying tag(s), thereby generating a further library of different bifunctional complexes comprising a reaction product and an identifier oligonucleotide comprising tags identifying the reactants which have participated in the formation of the reaction product.