Reactivation-based molecular interaction sensors

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.