Methods and compositions for selective labeling of different biotinylated targets within multicolor or multilabel assays
View Patent ↗Disclosed are compositions and methods for the labeling of two or more targets with different labels. Specifically, disclosed are compositions for biotin and the protection of biotin within multilabel assays which employ the biotin-biotin binding protein binding relationship for each distinct label in relation to targets such as nucleic acids, polypeptides, antibodies or cells. These multilabel assays are enabled through the use of biotin with desthiobiotin, orthogonal protecting schemes for biotin, or a combination of the approaches.
1. A method of labeling a plurality of targets with a plurality of labels, the method comprising:
providing a plurality of targets, wherein the plurality of targets comprises a first target and a second target, wherein the first target includes a first biotin molecule, and wherein the second target includes a second biotin molecule that is protected by a protecting group;
binding the first biotin molecule with a first biotin binding protein, wherein the first biotin binding protein includes a first label;
deprotecting the second biotin molecule, and
binding the deprotected second biotin molecule with a second biotin binding protein, wherein the second biotin binding protein includes a second label, and wherein the first label is distinguishable from the second label.
2. The method of claim 1 , wherein the plurality of targets was biotinylated to incorporate the first biotin molecule and the second biotin molecule.
3. A method of labeling a plurality of targets with a plurality of labels, the method comprising:
providing a plurality of targets, wherein the plurality of targets comprises a first target and a second target, wherein the first target includes a first biotin molecule, and wherein the second target includes a second biotin molecule, wherein the first biotin molecule has a stronger binding affinity for a biotin binding protein compared to the second biotin molecule;
binding the first biotin molecule and the second biotin molecule with a first biotin binding protein, wherein the first biotin binding protein includes a first label;
removing the first biotin binding protein from the second biotin molecule by introduction of a solution of the first biotin molecule, and
binding the second biotin molecule with a second biotin binding protein including a second label, and wherein the first label is different from the second label.
4. The method of claim 3 , wherein the first biotin molecule comprises biotin and the second biotin molecule comprises desthiobiotin.
5. The method of claim 1 , wherein at least one of either the first biotin binding protein or the second biotin binding protein is streptavidin.
6. The method of claim 1 , wherein at least one of either the first biotin binding protein or the second biotin binding protein is a recombinant protein.
7. The method of claim 1 , wherein the first biotin binding protein is a different protein than the second biotin binding protein.
8. The method of claim 1 , wherein the first label and the second label are fluorophores, wherein the first label has a first emission spectrum, wherein the second label has a second emission spectrum, and wherein the first emission spectrum is at least partially non-overlapping with the second emission spectrum.
9. The method of claim 1 , wherein the protecting group is connected to the second biotin molecule through an ether linkage.
10. The method of claim 9 , wherein the protecting group is 6-nitropiperonyloxymethyl.
11. The method of claim 1 , wherein the protecting group is dimethoxytrityl.
12. The method of claim 4 , wherein the plurality of targets additionally comprises a third target and a fourth target, wherein the third target includes a third biotin molecule protected by a first protecting group, wherein the fourth target includes a fourth biotin molecule protected by a second protecting group, and wherein the first protecting group and the second protecting group are different protecting groups.
13. The method of claim 12 , additionally comprising:
binding the third biotin molecule with a third biotin binding protein, wherein the third biotin binding protein includes a third label, and wherein the first protecting group is removed to allow the third biotin molecule to bind with the third biotin binding protein; and
binding the fourth biotin molecule with a fourth biotin binding protein, wherein the fourth biotin binding protein includes a fourth label, and wherein the second protecting group is removed to allow the fourth biotin molecule to bind with the fourth biotin binding protein.
14. The method of claim 12 , wherein the first biotin molecule is protected by a third protecting group, and wherein the second biotin molecule is protected by a fourth protecting group.
15. The method of claim 13 , wherein the first protecting group and the second protecting group are each removed through a distinct removal mechanism.