Botulinum neurotoxin-specific capture agents, compositions, and methods of using and making

1. A capture agent to a target produced by a method comprising the steps of:

(a) selecting a linker to connect an anchor ligand to a secondary ligand, wherein the anchor ligand and the secondary ligand bind to the same target at different binding sites representing distinct epitopes, wherein the linker is selected based on the distance between the binding site of the anchor ligand and the binding site of the secondary ligand,

wherein the anchor ligand and the secondary ligand bind their respective binding sites on the target in an orientation such that the target can promote 1,3-dipolar cycloaddition between (i) an acetylene group on the anchor ligand and an azide group on the secondary ligand or (ii) an azide group on the anchor ligand and an acetylene group on the secondary ligand to form a triazole linkage in the absence of a separate catalyst, wherein for the cycloaddition reaction, either the azide group or the acetylene group is connected to the anchor ligand or the secondary ligand via the selected linker;

(b) synthesizing a capture agent comprising the anchor ligand, the secondary ligand, and the selected linker, thereby generating the capture agent, wherein the dissociation constant of the capture agent for binding to the target is lower than the dissociation constant of either the anchor ligand or the secondary ligand for binding to the target.

2. The capture agent of claim 1 , wherein the linker has a length that is within 10% of the distance between the anchor ligand and the secondary ligand when both ligands are bound to the target protein.

3. The capture agent of claim 1 further comprising, prior to selecting the linker, the steps of:

(i) identifying an anchor ligand and a secondary ligand that bind to the same target peptide at distinct epitopes;

(ii) identifying the binding sites of the anchor ligand and the secondary ligand on the target peptide; and

(iii) calculating the distance between the binding site of the anchor ligand and the secondary ligand.

4. The capture agent of claim 1 , wherein the target is a protein.

5. The capture agent of claim 1 , wherein the anchor ligand is identified by binding to an epitope on the target.

6. The capture agent of claim 1 , wherein the secondary ligand is identified by binding to an epitope on the target.

7. The capture agent of claim 1 further comprising, prior to selecting the linker, identifying the anchor ligand, the secondary ligand, and the binding sites of the anchor ligand and the secondary capture agent on the target.

8. The capture agent of claim 1 further comprising, prior to selecting the linker, calculating the distance between the binding site of the anchor ligand and the binding site of the secondary ligand.

9. The capture agent of claim 1 , wherein the capture agent further comprises a tertiary ligand.

10. A capture agent targeting adjacent epitopes on a single protein produced by a method comprising

(a) identifying an anchor ligand that binds to a first epitope and a secondary ligand that binds to a second epitope, wherein the first and second epitopes are adjacent on the single protein;

(b) screening the anchor ligand and secondary ligand against a linker library based on affinity of the combined anchor ligand, linker and secondary ligand to bind the single protein; and

(c) selecting a linker to connect the anchor ligand to the secondary ligand, thereby targeting adjacent epitopes of the single protein by generating a capture agent that targets adjacent epitopes on the single protein, wherein the dissociation constant of the capture agent for binding to the target protein is lower than the dissociation constant of either the anchor ligand or the secondary ligand for binding to the single protein.

11. The method of claim 10 , wherein the adjacent epitopes are 5-10 angstroms apart on the single protein when the single protein is folded.