Methods and Compositions for Selective Labeling of Different Biotinylated Targets within Multicolor or Multilabel Assays

Abstract

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.

What is claimed is: 
     
         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;   binding the first biotin molecule with a first biotin binding protein, wherein the first biotin binding protein includes a first label; and   binding the second biotin molecule with a second biotin binding protein, wherein the second biotin binding protein includes a second label.   
     
     
         2 . The method of  claim 1 , wherein the plurality of targets comprises polynucleotides. 
     
     
         3 . The method of  claim 1 , wherein the plurality of targets comprises polypeptides. 
     
     
         4 . The method of  claim 1 , wherein the plurality of targets comprises antibodies. 
     
     
         5 . The method of  claim 1 , wherein the plurality of targets comprises cells. 
     
     
         6 . The method of  claim 1 , wherein the plurality of targets was biotinylated to incorporate the first biotin molecule and the second biotin molecule. 
     
     
         7 . The method of  claim 1 , wherein the plurality of targets additionally comprises a third target and a fourth target, wherein the third target includes a third biotin molecule, and wherein the fourth target includes a fourth biotin molecule. 
     
     
         8 . The method of  claim 1 , wherein the first biotin molecule and the second biotin molecule comprise biotin molecules with distinct binding affinities for biotin binding proteins. 
     
     
         9 . The method of  claim 8 , wherein the first biotin molecule comprises biotin and the second biotin molecule comprises desthiobiotin. 
     
     
         10 . The method of  claim 1 , wherein at least one of either the first biotin binding protein or the second biotin binding protein is streptavidin. 
     
     
         11 . 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. 
     
     
         12 . The method of  claim 1 , wherein the first biotin binding protein is a different protein than the second biotin binding protein. 
     
     
         13 . The method of  claim 1 , wherein the first label is distinguishable from the second label. 
     
     
         14 . The method of  claim 13 , 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. 
     
     
         15 . The method of  claim 1 , wherein the second biotin molecule is protected by a protecting group. 
     
     
         16 . The method of  claim 15 , wherein the protecting group is a photolabile protecting group. 
     
     
         17 . The method of  claim 15 , wherein the protecting group is an acid-labile protecting group. 
     
     
         18 . The method of  claim 15 , wherein the protecting group is a base-labile protecting group. 
     
     
         19 . The method of  claim 15 , wherein the protecting group is connected to the second biotin molecule through an ether linkage. 
     
     
         20 . The method of  claim 19 , wherein the protecting group is 6-nitropiperonyloxymethyl. 
     
     
         21 . The method of  claim 17 , wherein the protecting group is dimethoxytrityl. 
     
     
         22 . The method of  claim 15 , wherein binding the second biotin molecule with the second biotin binding protein comprises:
 removing the protecting group from the second biotin molecule; and   binding the second biotin binding protein to the second biotin molecule.   
     
     
         23 . The method of  claim 22 , wherein the first biotin molecule is protected by a second protecting group, and wherein binding the first biotin molecule with the first biotin binding protein comprises:
 removing the second protecting group from the first biotin molecule; and   binding the first biotin binding protein to the first biotin molecule.   
     
     
         24 . The method of  claim 22 , wherein the plurality of targets additionally comprises a third target and a fourth target, wherein the third target includes a third biotin molecule, wherein the fourth target includes a fourth biotin molecule, wherein the third biotin molecule is protected by a second protecting group, wherein the fourth biotin molecule is protected by a third protecting group, and wherein the protecting group of the second biotin molecule, the second protecting group and the third protecting group are each different protecting groups. 
     
     
         25 . The method of  claim 24 , 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 second 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 third protecting group is removed to allow the fourth biotin molecule to bind with the fourth biotin binding protein.   
     
     
         26 . The method of  claim 25 , wherein the protecting group of the second biotin molecule, the second protecting group and the third protecting group are each removed through a distinct removal mechanism. 
     
     
         27 . The method of  claim 26 , wherein the distinct removal mechanism includes removal of at least one type of protecting group through radiation comprising one or more selected wavelengths. 
     
     
         28 . The method of  claim 26 , wherein the distinct removal mechanism includes removal of at least one type of protecting group through an acid treatment. 
     
     
         29 . The method of  claim 28 , wherein the acid treatment comprises treatment with an acid solution containing an acid selected from the group consisting of trifluoroacetic acid, acetic acid and monopotassium phosphate. 
     
     
         30 . The method of  claim 26 , wherein the distinct removal mechanism includes removal of at least one type of protecting group through a base treatment. 
     
     
         31 . The method of  claim 9 , wherein binding the desthiobiotin with the second biotin binding protein comprises:
 displacing the desthiobiotin from the first biotin binding protein with a biotin solution; and   binding the desthiobiotin with the second biotin binding protein.   
     
     
         32 . The method of  claim 31 , 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. 
     
     
         33 . The method of  claim 32 , 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.   
     
     
         34 . The method of  claim 32 , 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. 
     
     
         35 . The method of  claim 33 , wherein the first protecting group and the second protecting group are each removed through a distinct removal mechanism. 
     
     
         36 . The method of  claim 35 , wherein the distinct removal mechanism includes removal of at least one type of protecting group through radiation comprising one or more selected wavelengths. 
     
     
         37 . The method of  claim 35 , wherein the distinct removal mechanism includes removal of at least one type of protecting group through an acid treatment. 
     
     
         38 . The method of  claim 37 , wherein the acid treatment comprises treatment with an acid solution containing an acid selected from the group consisting of trifluoroacetic acid, acetic acid and monopotassium phosphate. 
     
     
         39 . The method of  claim 35 , wherein the distinct removal mechanism includes removal of at least one type of protecting group through a base treatment. 
     
     
         40 . 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, a second target, a third target and a fourth target, wherein the first target includes a first biotin molecule, wherein the second target includes a second biotin molecule protected by a first protecting group, wherein the third target includes a third biotin molecule protected by a second protecting group, and wherein the fourth target includes a fourth biotin molecule protected by a third protecting group;   binding the first biotin molecule with a first biotin binding protein, wherein the first biotin binding protein includes a first label;   binding the second biotin molecule with a second biotin binding protein, wherein the second biotin binding protein includes a second label, and wherein the first protecting group is removed to allow the second biotin molecule to bind with the second biotin binding protein;   binding the third biotin molecule with a third biotin binding protein, wherein the third biotin binding protein includes a third label, and wherein the second 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 third protecting group is removed to allow the fourth biotin molecule to bind with the fourth biotin binding protein.   
     
     
         41 . 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, a second target, a third target and a fourth target, wherein the first target includes a first biotin molecule, wherein the first biotin molecule comprises biotin, wherein the second target includes a second biotin molecule, wherein the second biotin molecule comprises desthiobiotin, wherein the third target includes a third biotin molecule protected by a first protecting group, and wherein the fourth target includes a fourth biotin molecule protected by a second protecting group;   binding the first biotin molecule with a first biotin binding protein, wherein the first biotin binding protein includes a first label, and wherein binding the first biotin molecule with the first biotin binding protein also binds the second biotin molecule with the first biotin binding protein;   binding the second biotin molecule with a second biotin binding protein, wherein the second biotin binding protein includes a second label, and wherein the first biotin binding protein is displaced from the second biotin molecule with a biotin solution;   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.