Capture agents and related compositions, methods and systems
The present disclosure relates to capture agents for the detection and/or separation of one or more targets in a sample. Described herein are multi-ligand capture agents comprising two or more ligands, and related compositions, methods and systems. In certain embodiments, the capture agents disclosed herein can be used for performing assays, including but not limited to assays for the detection and/or separation of targets.
1. A method of making higher-order multi-ligand capture agents, the method comprising:
contacting a modified anchor ligand with a first plurality of candidate ligands and a molecular target, wherein the first plurality of candidate ligands comprises at least about 3200 unique ligands,
whereby the modified anchor ligand is covalently linked to one or more of the candidate ligands in a reaction catalyzed by the molecular target, thereby forming one or more bi-ligand capture agents, wherein each of the bi-ligand capture agents comprises the modified anchor ligand and one of the candidate ligands,
wherein the anchor ligand can bind the molecular target, wherein the modified anchor ligand comprises a first functional group, wherein the first functional group is capable of specifically reacting with a corresponding second functional group, wherein each of the candidate ligands comprises the second functional group;
modifying one of the bi-ligand capture agents by covalently attaching a third functional group, wherein the third functional group is capable of specifically reacting with a corresponding fourth functional group, and
contacting the modified bi-ligand capture agent with a second plurality of candidate ligands and the molecular target, wherein the second plurality of candidate ligands comprises at least about 3200 unique ligands,
whereby the modified bi-ligand capture agent is covalently linked to one or more of the candidate ligands in a reaction catalyzed by the molecular target, thereby forming one or more higher order tri-ligand capture agents, wherein each of the higher order tri-ligand capture agents comprises the modified bi-ligand capture agent and one of the candidate ligands,
wherein each of the candidate ligands in the second plurality of capture ligands comprises the fourth functional group, wherein the plurality of candidate ligands comprises a combinatorial library of compounds, wherein the combinatorial library of compounds comprises compounds comprising permutations of a group of subunits linked in chains; and
wherein the dissociation constant for binding of the higher-order tri-ligand capture agent to the molecular target is smaller than the dissociation constant for binding of the bi-ligand capture agent to the molecular target, the dissociation constant for binding of the bi-ligand capture agent to the molecular target is smaller than the dissociation constant for binding of the anchor ligand to the molecular target, wherein the dissociation constant of the tri-ligand capture agent to the molecular target is at least 1×10 −6 and wherein the anchor ligand, the candidate ligand covalently linked to the anchor ligand and the candidate ligand covalently linked to the bi-ligand bind to the molecular target at distinct epitopes.
2. The method of claim 1 , wherein prior to contacting it is not known which of the candidate ligands can bind to the molecular target.
3. The method of claim 1 , wherein the chains are straight, branched, circular, or a combination.
4. The method of claim 1 , wherein the group of subunits comprises amino acids, modified amino acids, or a combination.
5. The method of claim 4 , wherein the group of subunits further comprises one or more small organic molecules.
6. The method of claim 4 , wherein the amino acids in the group of subunits consists essentially of a subset of amino acids.
7. The method of claim 4 , wherein the modified amino acids in the group of subunits consists essentially of a subset of modified amino acids.
8. The method of claim 1 , wherein the first plurality of candidate ligands comprises at least about 117,649 unique ligands and wherein the second plurality of candidate ligands comprises at least about 117,649 unique ligands.
9. The method of claim 1 , wherein the first plurality of candidate ligands comprises at least about 262,144 unique ligands and wherein the second plurality of candidate ligands comprises at least about 262,144 unique ligands.
10. The method of claim 1 , wherein the first plurality of candidate ligands comprises at least about 2,476,099 unique ligands and wherein the second plurality of candidate ligands comprises at least about 2,476,099 unique ligands.