Reactivation-based molecular interaction sensors

• US 20030198971A1
• Filed: 07/29/2002
• Published: 10/23/2003
• Est. Priority Date: 04/18/2002
• Status: Active Grant

• Current Assignee: Kalobios Incorporated
• Original Assignee: Kalobios Incorporated
• Inventors: Her, Jeng-Horng, Balint, Robert F.
• Granted Patent: US 7,335,478 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

This invention provides methods and systems for linking the functional activation of a responder molecule to the interaction of two or more binding ensemble members of interest either in vitro or in vivo, and thereby producing a signal, phenotype, or other functional output in response to the interaction of the binding ensemble members. The systems comprise a responder, an inhibitor of the responder, and an inhibitor of the inhibitor, or “reactivator” of the responder, and interacting components. Two binding ensemble members are complexed to the other components of the system in such a way that interaction of the binding ensemble members, either directly or via additional ensemble members, causes a shift in the equilibrium of inhibitor binding from the responder to the reactivator, thereby functionally activating the responder.

Claims

1. A method for detecting an interaction of binding ensemble members, comprising the steps of:

providing a responder complex comprising a responder molecule, an inhibitor of the responder molecule, and a first member of a binding ensemble;

providing a reactivator complex comprising a reactivator molecule and a second member of the binding ensemble;

combining the responder complex and the reactivator complex; and

detecting an activity of the responder molecule, thereby detecting the interaction of the first and the second binding ensemble members.

2. The method of claim 1, wherein the first member of the binding ensemble is linked to the responder molecule and the inhibitor is linked to the responder molecule.

3. The method of claim 2, wherein the first member of the binding ensemble and the responder molecule are linked by a first covalent linkage;

the inhibitor and the responder molecule are linked by a second covalent linkage; and

further, wherein the reactivator and the second member of the binding ensemble by a third covalent linkage.

4. The method of claim 2, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

5. The method of claim 2, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

6. The method of claim 2, further comprising a third member of the binding ensemble, wherein the first member of the binding ensemble and the second member of the binding ensemble interact with the third member of the binding ensemble.

7. The method of claim 6, wherein the third member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

8. The method of claim 6, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

9. The method of claim 6, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

10. The method of claim 6, wherein the third member of the binding ensemble is a member of a plurality of candidate binding ensemble members;

the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members; and

the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

11. The method of claim 1, wherein the responder molecule is linked to the first member of the binding ensemble, and the inhibitor is linked to the first member of the binding ensemble.

12. The method of claim 11, wherein the first member of the binding ensemble and the responder molecule are linked by a first covalent linkage, the inhibitor and the first member of the binding ensemble are linked by a second covalent linkage;

and further, wherein the reactivator and the second member of the binding ensemble are linked by a third covalent linkage.

13. The method of claim 12, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

14. The method of claim 12, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

15. The method of claim 12, further comprising a third member of the binding ensemble, wherein the first member of the binding ensemble and the second member of the binding ensemble interact with the third member of the binding ensemble.

16. The method of claim 15, wherein the third member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

17. The method of claim 15, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

18. The method of claim 15, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

19. The method of claim 15, wherein the third member of the binding ensemble is a member of a plurality of candidate binding ensemble members;

and further, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members and the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

20. The method of claim 1, wherein the responder molecule is linked to the inhibitor, and the first member of the binding ensemble is linked to the inhibitor.

21. The method of claim 20, wherein the responder molecule and the inhibitor are linked by a first covalent linkage, the first member of the binding ensemble and the inhibitor are linked by a second covalent linkage, and further, wherein the reactivator and the second member of the binding ensemble are linked by a third covalent linkage.

22. The method of claim 20, wherein the responder molecule and the inhibitor are linked by a non-covalent linkage, the first member of the binding ensemble and the inhibitor are linked by a covalent linkage, and further, wherein the reactivator and the second member of the binding ensemble are linked by a covalent linkage.

23. The method of claim 21, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

24. The method of claim 21, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

25. The method of claim 21, further comprising a third member of the binding ensemble, wherein the first member of the binding ensemble and the second member of the binding ensemble interact with the third member of the binding ensemble.

26. The method of claim 25, wherein the third member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

27. The method of claim 25, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

28. The method of claim 25, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

29. The method of claim 25, wherein the third member of the binding ensemble is a member of a plurality of candidate binding ensemble members;

and further, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members and the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

30. A responder complex comprising a responder molecule, an inhibitor of the responder molecule, and a binding ensemble member;

wherein the binding ensemble member is linked to the responder molecule and the inhibitor is linked to the responder molecule.

31. The responder complex of claim 30, wherein the binding ensemble member and the responder molecule are linked by a first covalent linkage, and the inhibitor and the responder molecule are linked by a second covalent linkage.

32. The responder complex of claim 30, wherein the binding ensemble member and the responder molecule are linked by a first non-covalent linkage, and the inhibitor and the responder molecule are linked by a second non-covalent linkage.

33. The responder complex of claim 30, wherein the binding ensemble member and the responder molecule are linked by a covalent linkage, and the inhibitor and the responder molecule are linked by a non-covalent linkage.

34. The responder complex of claim 30, wherein the binding ensemble member and the responder molecule are linked by a non-covalent linkage, and the inhibitor and the responder molecule are linked by a covalent linkage.

35. The responder complex of claim 30, wherein the binding ensemble member is a member of a plurality of candidate binding ensemble members.

36. The responder complex of claim 31, wherein the first covalent linkage comprises a linker.

37. The responder complex of claim 31, where the second covalent linkage comprises a linker.

38. A nucleic acid encoding the responder complex of claim 31.

39. An expression vector comprising the nucleic acid of claim 38 operatively linked to a control region.

40. A host cell comprising an expression vector of claim 39.

41. A system comprising:

a responder complex comprising a responder molecule, an inhibitor, and a first member of a binding ensemble;

wherein the first member of the binding ensemble is linked to the responder molecule and the inhibitor is linked to the responder molecule; and

a reactivator complex comprising a reactivator molecule and a second member of the binding ensemble, wherein the reactivator molecule is linked to the second member of the binding ensemble.

42. The system of claim 41, wherein the first member of the binding ensemble and the responder molecule are linked by a first covalent linkage, the inhibitor and the responder molecule are linked by a second covalent linkage, and the reactivator and the second member of the binding ensemble are linked by a third covalent linkage.

43. The system of claim 41, wherein the first member of the binding ensemble and the responder molecule are linked by a first non-covalent linkage, the inhibitor and the responder molecule are linked by a second non-covalent linkage, and the reactivator and the second member of the binding ensemble are linked by a third non-covalent linkage.

44. The system of claim 42, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

45. The system of claim 42, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

46. The system of claim 42, further comprising a third member of the binding ensemble, wherein the first member of the binding ensemble and the second member of the binding ensemble interact with the third member of the binding ensemble.

47. The system of claim 46, wherein the third member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

48. The system of claim 47, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

49. The system of claim 47, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

50. The system of claim 47, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members and the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

51. A host cell comprising the system of claim 42.

52. A reactivator complex comprising a reactivator molecule and a binding ensemble member, wherein the reactivator molecule is linked to the binding ensemble member.

53. The reactivator complex of claim 52, wherein the reactivator molecule and the binding ensemble member are linked by a covalent linkage.

54. The reactivator complex of claim 52, wherein the reactivator molecule and the binding ensemble member are linked by a noncovalent linkage.

55. The reactivator complex of claim 53, wherein the binding ensemble member is a member of a plurality of candidate binding ensemble members.

56. The reactivator complex of claim 53, wherein the covalent linkage comprises a linker.

57. A nucleic acid encoding the reactivator complex of claim 53.

58. An expression vector comprising the nucleic acid of claim 57 operatively linked to a control region.

59. A host cell comprising an expression vector of claim 58.

60. A host cell comprising the reactivator complex of claim 53.

85. The responder complex of claim 55, wherein the responder molecule and the inhibitor are linked by a first non-covalent linkage, and the binding ensemble member and the inhibitor are linked by a second non-covalent linkage.

87. The responder complex of claim 55, wherein the responder molecule and the inhibitor are linked by a covalent linkage, and the binding ensemble member and the inhibitor are linked by a non-covalent linkage.

61. A responder complex comprising a responder molecule, an inhibitor of the responder molecule, and a binding ensemble member;

wherein the responder molecule is linked to the binding ensemble member and the inhibitor is linked to the binding ensemble member.

62. The responder complex of claim 61, wherein the responder molecule and the binding ensemble member are linked by a first covalent linkage, and the inhibitor and the binding ensemble member are linked by a second covalent linkage.

63. The responder complex of claim 61, wherein the responder molecule and the binding ensemble member are linked by a first non-covalent linkage, and the inhibitor and the binding ensemble member are linked by a second non-covalent linkage.

64. The responder complex of claim 61, wherein the responder molecule and the binding ensemble member are linked by a non-covalent linkage, and the inhibitor and the binding ensemble member are linked by a covalent linkage.

65. The responder complex of claim 61, wherein the responder molecule and the binding ensemble member are linked by a covalent linkage, and the inhibitor and the binding ensemble member are linked by a non-covalent linkage.

66. The responder complex of claim 62, wherein the binding ensemble member is a member of a plurality of candidate binding ensemble members.

67. The responder complex of claim 62, wherein the first covalent linkage comprises a linker.

68. The responder complex of claim 62, wherein the second covalent linkage comprises a linker.

69. A nucleic acid encoding the responder complex of claim 62.

70. An expression vector comprising the nucleic acid of claim 69 operatively linked to a control region.

71. A host cell comprising an expression vector of claim 70.

72. A system comprising:

a responder complex comprising a responder molecule, an inhibitor, and a first member of a binding ensemble;

wherein the responder molecule is linked to the first binding ensemble member and the inhibitor is linked to the first member of the binding ensemble; and

a reactivator complex comprising a reactivator molecule and a second member of the binding ensemble, wherein the reactivator molecule is linked to the second member of the binding ensemble.

73. The system of claim 72, wherein the first member of the binding ensemble and the responder molecule are linked by a first covalent linkage;

the inhibitor and the first binding ensemble member are linked by a second covalent linakge; and

the reactivator and the second member of the binding ensemble are linked by a third covalent linkage.

74. The system of claim 72, wherein the first member of the binding ensemble and the responder molecule are linked by a first non-covalent linkage;

the inhibitor and the first binding ensemble member are linked by a second non-covalent linakge; and

the reactivator and the second member of the binding ensemble are linked by a third non-covalent linkage.

75. The system of claim 73, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

76. The system of claim 73, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

77. The system of claim 73, further comprising a third member of the binding ensemble, wherein the first member of the binding ensemble and the second member of the binding ensemble interact with the third member of the binding ensemble.

78. The system of claim 77, wherein the third member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

79. The system of claim 78, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

80. The system of claim 78, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

81. The system of claim 78, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members and the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

82. A host cell comprising the system of claim 73.

83. A responder complex comprising a responder molecule, an inhibitor of the responder molecule, and a binding ensemble member;

wherein the responder molecule is linked to the inhibitor and the binding ensemble member is linked to the inhibitor.

84. The responder complex of claim 83, wherein the responder molecule and the inhibitor are linked by a first covalent linkage, and the binding ensemble member and the inhibitor are linked by a second covalent linkage.

86. The responder complex of claim 83, wherein the responder molecule and the inhibitor are linked by a non-covalent linkage, and the binding ensemble member and the inhibitor are linked by a covalent linkage.

88. The responder complex of claim 84, wherein the binding ensemble member is a member of a plurality of candidate binding ensemble members.

89. The responder complex of claim 84, wherein the first covalent linkage comprises a linker.

90. The responder complex of claim 84, wherein the second covalent linkage comprises a linker.

91. A nucleic acid encoding the responder complex of claim 84.

92. An expression vector comprising the nucleic acid of claim 91 operatively linked to a control region.

93. A host cell comprising an expression vector of claim 92.

94. A system comprising:

a responder complex comprising a responder molecule, an inhibitor, and a first member of a binding ensemble;

wherein the responder is linked to the inhibitor and the first member of the binding ensemble is linked to the inhibitor; and

a reactivator complex comprising a reactivator molecule and a second member of the binding ensemble, wherein the reactivator molecule is linked to the second member of the binding ensemble.

95. The system of claim 94, wherein the responder molecule and the inhibitor are linked by a first covalent linkage, the first member of the binding ensemble and the inhibitor are linked by a second covalent linkage;

and the reactivator and the second member of the binding ensemble are linked by a third covalent linkage.

96. The system of claim 94, wherein the responder molecule and the inhibitor are linked by a first non-covalent linkage, the first member of the binding ensemble and the inhibitor are linked by a second non-covalent linkage;

and the reactivator and the second member of the binding ensemble are linked by a third non-covalent linkage.

97. The system of claim 94, wherein the responder molecule and the inhibitor are linked by a non-covalent linkage, the first member of the binding ensemble and the inhibitor are linked by a covalent linkage;

and further, wherein the reactivator and the second member of the binding ensemble are linked by a covalent linkage.

98. The system of claim 95, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

99. The system of claim 95, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

100. The system of claim 95, further comprising a third member of the binding ensemble, wherein the first member of the binding ensemble and the second member of the binding ensemble interact with the third member of the binding ensemble.

101. The system of claim 100, wherein the third member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

102. The system of claim 100, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

103. The system of claim 100, wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

104. The system of claim 100, wherein the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members;

and wherein the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.

105. A host cell comprising the system of claim 95.

106. A method of interaction mapping, comprising the steps of:

providing a plurality of responder complexes comprising a responder molecule, an inhibitor and a binding ensemble member;

providing a plurality of reactivator complexes comprising a plurality of candidate binding ensemble members, wherein each candidate binding ensemble member is individually linked to a reactivator molecule;

individually combining at least one member of the plurality of reactivator complexes with a responder complex; and

detecting an activity of the responder molecule, thereby detecting the interaction of the binding ensemble member with at least one of the plurality of candidate binding ensemble members.

107. The method of claim 106, wherein the binding ensemble member is linked to the responder molecule and the inhibitor is linked to the responder molecule.

108. The method of claim 107, wherein the binding ensemble member and the responder molecule are linked by a first covalent linkage, the inhibitor and the responder molecule are linked by a second covalent linkage;

and further, wherein the reactivator molecule and the candidate binding ensemble members are linked by a third covalent linkage.

109. The method of claim 108, wherein the plurality of responder complexes comprise two or more different candidate binding ensemble members.

110. The method of claim 106, wherein the responder molecule is linked to the binding ensemble member and the inhibitor is linked to the binding ensemble member.

111. The method of claim 110, wherein the binding ensemble member and the responder molecule are linked by a first covalent linkage, the inhibitor and the binding ensemble member are linked by a second covalent linkage, and further, wherein the reactivator molecule and the candidate binding ensemble member are linked by a third covalent linkage.

112. The method of claim 111, wherein the plurality of responder complexes comprise two or more different candidate binding ensemble members.

113. The method of claim 106, wherein the responder molecule is linked to the inhibitor and the binding ensemble member is linked to the inhibitor.

114. The method of claim 113, wherein the responder molecule and the inhibitor are linked by a first covalent linkage, the binding ensemble member and the inhibitor are linked by a second covalent linkage;

and further, wherein the reactivator molecule and the candidate binding ensemble members are linked by a third covalent linkage.

115. The method of claim 114, wherein the plurality of responder complexes comprise two or more different candidate binding ensemble members.

116. A method of interaction mapping, comprising the steps of:

providing a plurality of responder complexes comprising a plurality of candidate binding ensemble members;

providing a plurality of reactivator complexes comprising a binding ensemble member linked to a reactivator molecule;

combining the plurality of responder complexes with the plurality of reactivator complexes; and

detecting an activity of the responder molecule, thereby detecting interaction between the binding ensemble member and at least one of the plurality of candidate binding ensemble members.

117. The method of claim 116, wherein the candidate binding ensemble member is linked to the responder molecule and the inhibitor is linked to the responder molecule.

118. The method of claim 117, wherein the candidate binding ensemble member and the responder molecule are linked by a first covalent linkage, the inhibitor and the responder molecule is a second covalent linkage;

and further, wherein the reactivator and the binding ensemble member are linked by a third covalent linkage.

119. The method of claim 118, wherein the plurality of reactivator complexes comprise two or more different candidate binding ensemble members.

120. The method of claim 116, wherein the responder molecule is linked to the candidate binding ensemble member and the inhibitor is linked to the candidate binding ensemble member.

121. The method of claim 120, wherein the candidate binding ensemble member and the responder molecule are linked by a first covalent linkage, the inhibitor and the candidate binding ensemble member are linked by a second covalent linkage, and further wherein the reactivator and the binding ensemble member are linked by a third covalent linkage.

122. The method of claim 121, wherein the plurality of reactivator complexes comprise two or more different candidate binding ensemble members.

123. The method of claim 116, wherein the responder molecule is linked to the inhibitor and the candidate binding ensemble member is linked to the inhibitor.

124. The method of claim 123, wherein the responder molecule and the inhibitor are linked by a first covalent linkage, the candidate binding ensemble member and the inhibitor are linked by a second covalent linkage;

and further, wherein the reactivator and the binding ensemble member are linked by a third covalent linkage.

125. The method of claim 124, wherein the plurality of reactivator complexes comprise two or more different candidate binding ensemble members.

126. A method for improving the affinity of a first binding pair member, the method comprising the steps of:

providing a plurality of a first binding pair member;

providing a plurality of a reactivator complex comprising a reactivator molecule and a second binding pair member;

providing a plurality of responder complexes comprising a plurality of a responder molecule, a plurality of an inhibitor, and a plurality of candidate binding pair members, wherein the plurality of candidate binding pair members comprises variants of the first binding pair member;

combining the reactivator complexes, the responder complexes, and the plurality of the first binding pair member, wherein a responder molecule is activated when a candidate binding pair member binds to a second binding pair member; and

wherein the activity of the responder molecule is proportional to the affinity of the candidate binding pair members for the second binding pair member; and

detecting an activity of the responder molecule corresponding to an affinity of the candidate binding pair member for the second binding pair member that is greater than that of the first binding pair member for the second binding pair member.

127. The method of claim 126, wherein the candidate binding ensemble member is linked to the responder molecule and the inhibitor is linked to the responder molecule.

128. The method of claim 127, wherein the candidate binding ensemble member and the responder molecule are linked by a first covalent linkage, the inhibitor and the responder molecule are linked by a second covalent linkage, and further, wherein the reactivator and the first member of the binding ensemble are linked by a third covalent linkage.

129. The method of claim 126, wherein the responder molecule is linked to the candidate binding ensemble member and the inhibitor is linked to the candidate binding ensemble member.

130. The method of claim 129, wherein the candidate binding ensemble member and the responder molecule are linked by a first covalent linkage, the inhibitor and the candidate binding ensemble member are linked by a second covalent linkage, and further, wherein the reactivator and the first member of the binding ensemble are linked by a third covalent linkage.

131. The method of claim 126, wherein the responder molecule is linked to the inhibitor and the candidate binding ensemble member is linked to the inhibitor.

132. The method of claim 131, wherein the responder molecule and the inhibitor are linked by a first covalent linkage, the candidate binding ensemble member and the inhibitor are linked by a second covalent linkage;

and further, wherein the reactivator and the first member of the binding ensemble are linked by a third covalent linkage.

133. A method for improving the affinity of a first binding pair member, the method comprising the steps of:

providing a plurality of a first binding pair member;

providing a plurality of a responder complex comprising a responder molecule, an inhibitor, and a second binding pair member;

providing a plurality of reactivator complexes comprising a plurality of a reactivator molecule and a plurality of candidate binding pair members, wherein the plurality of candidate binding pair members comprise variants of the first binding pair member;

combining the reactivator complexes, the responder complexes, and the plurality of the first binding pair member, wherein a responder molecule is activated when a candidate binding pair member binds to a second binding pair member; and

wherein the activity of the responder molecule is proportional to the affinity of the candidate binding pair member for the second binding pair member; and

detecting an activity of the responder molecule corresponding to an affinity of the candidate binding pair member for the second binding pair member that is greater than that of the first binding pair member for the second binding pair member.

134. The method of claim 133, wherein the first member of the binding ensemble is linked to the responder molecule and the inhibitor is linked to the responder molecule.

135. The method of claim 134, wherein the first member of the binding ensemble and the responder molecule are linked by a first covalent linkage, the inhibitor and the responder molecule are linked by a second covalent linkage;

and further, wherein the reactivator and the candidate binding ensemble are linked by a third covalent linkage.

136. The method of claim 133, wherein the responder molecule is linked to the first member of the binding ensemble and the inhibitor is linked to the first member of the binding ensemble.

137. The method of claim 136, wherein the first member of the binding ensemble and the responder molecule are linked by a first covalent linkage, the inhibitor and the first member of the binding ensemble are linked by a second covalent linkage, and further, wherein the reactivator and the candidate binding ensemble are linked by a third covalent linkage.

138. The method of claim 133, wherein the responder molecule is linked to the inhibitor and the first member of the binding ensemble is linked to the inhibitor.

139. The method of claim 138, wherein the responder molecule and the inhibitor are linked by a first covalent linkage, the first member of the binding ensemble and the inhibitor are linked by a second covalent linkage, and further, wherein the reactivator and the candidate binding ensemble are linked by a third covalent linkage.

140. A method for isotropic selection of a plurality of binding molecules, comprising the steps of:

providing a plurality of responder complexes comprising a responder molecule, an inhibitor, and a first member of a binding ensemble;

providing a first plurality of reactivator complexes comprising a plurality of candidate binding ensemble members, wherein each candidate binding ensemble member is individually linked to a reactivator molecule;

combining the plurality of responder complexes with the plurality of reactivator complexes;

detecting a first set of binding molecules, each of which binds to the first member of the binding ensemble, by detecting a responder activity when one of the candidate binding molecules bind to the first member of the binding ensemble;

providing a plurality of responder complexes, each comprising a responder, an inhibitor, and a binding molecule from the first set of binding molecules;

providing a second plurality of reactivator complexes comprising a plurality of candidate binding ensemble members, wherein each candidate binding ensemble member is individually linked to a reactivator molecule;

providing a plurality of the first member of the binding ensemble;

combining the plurality of responder complexes, the plurality of reactivator complexes, and the plurality of the first member of the binding ensemble; and

detecting the responder activity when the binding molecule from the first set of binding molecules and a candidate binding molecule are bound simultaneously to the first member of the binding ensemble.

141. The method of claim 140, wherein the first plurality of reactivator complexes and the second plurality of the reactivator complexes are the same.

142. A method for isotropic selection of a plurality of binding molecules, comprising the steps of:

providing a first plurality of responder complexes comprising a plurality of candidate binding ensemble members wherein each candidate binding ensemble member is individually mixed with a responder molecule, and an inhibitor;

providing a plurality of reactivator complexes comprising a reactivator linked to a first member of a binding ensemble;

combining the first plurality of responder complexes with the plurality of reactivator complexes;

detecting a first set of binding molecules, each of which binds to the first member of the binding ensemble, by detecting a responder activity when the candidate binding molecules bind to the first member of the binding ensemble;

providing a second plurality of responder complexes comprising a plurality of candidate binding ensemble members wherein each candidate binding ensemble member is individually mixed with a responder molecule, and an inhibitor;

providing a plurality of reactivator complexes, each comprising a reactivator linked to a binding molecule from the first set of binding molecules;

providing the plurality of the first member of the binding ensemble;

combining the second plurality of responder complexes, the plurality of reactivator complexes, and the plurality of the first member of the binding ensemble; and

detecting the responder activity when the binding molecule from the first set of binding molecules and a candidate binding molecule are bound simultaneously to the first member of the binding ensemble.

143. The method of claim 142, wherein the first plurality of responder complexes and the second plurality of the responder complexes are the same.

144. A method for isotropic selection of a plurality of binding molecules, comprising the steps of:

providing a plurality of responder complexes comprising a plurality of candidate binding ensemble members wherein each candidate binding ensemble member is individually linked to the responder molecule and the inhibitor;

providing a plurality of reactivator complexes comprising a plurality of candidate binding ensemble members, wherein each candidate binding ensemble member is individually linked to a reactivator molecule;

providing a plurality of a first member of a binding ensemble;

combining the plurality of responder complexes, the plurality of reactivator complexes and the plurality of the first member of the binding ensemble, wherein each combination is comprised of one responder complex, one reactivator complex, and the first binding ensemble member; and

detecting a responder activity when a binding molecule from the first set of candidate binding molecules and a binding molecule from the second set of candidate binding molecules are bound simultaneously to the first member of the binding ensemble.

145. The method of claim 144, wherein the first set of candidate binding molecules and the second set of candidate binding molecules are the same.