Bis-biotinylation tags

Abstract

Multi-biotinylated reactants are provided which can be used in divalent complexes for various applications such as colocalization, labeling, immobilization, and purification. Methods for constructing, purifying, and using the bis-biotinylated reactants are also provided. In certain embodiments, two bis-biotinylated reactants are bound to a single streptavidin tetramer to provide a complex having a 1:1 stoichiometry with respect to the bis-biotinylated reactants.

1 - 31 . (canceled) 
     
     
         32 . A compound having the formula 
       
         
           
           
               
               
           
         
       
       wherein S comprises a silane moiety, PEG comprises polyethylene glycol, C is a core group, L 3  is a direct bond or a first linking group, L 4  is a direct bond or a second linking group, and B is a biotin group. 
     
     
         33 . The compound of  claim 32 , wherein L 3  is a linking group comprising a negatively charged group and L 4  is a linking group comprising a negatively charged group. 
     
     
         34 . The compound of  claim 33 , wherein L 3  and L 4  comprise a sulfonic acid moiety. 
     
     
         35 . The compound of  claim 33 , wherein L 3  and L 4  comprise a methylsulfonic acid group. 
     
     
         36 . The compound of  claim 33 , wherein L 3  and L 4  each comprise three sulfonic acid moieties. 
     
     
         37 . The compound of  claim 32 , having the formula 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein n is between 2 and 250. 
     
     
         38 . A method of preparing a surface, the method comprising coating the surface with the compound of  claim 32 , wherein reaction of the compound with the surface covalently couples the two biotin groups to the surface. 
     
     
         39 - 41 . (canceled) 
     
     
         42 . The method of  claim 38 , wherein the surface comprises the bottom surface of at least one nanoscale well. 
     
     
         43 . The method of  claim 38 , comprising binding a tetravalent biotin-binding agent to the two biotin groups. 
     
     
         44 . The method of  claim 43 , comprising binding a molecule or molecular complex of interest to the tetravalent biotin-binding agent. 
     
     
         45 . The method of  claim 44 , wherein binding the molecule or molecular complex of interest to the tetravalent biotin-binding agent is performed before binding the tetravalent biotin-binding agent to the two biotin groups. 
     
     
         46 . The method of  claim 44 , wherein a bis-biotin tag is covalently coupled to the molecule or molecular complex of interest, and wherein the bis-biotin tag is bound to the tetravalent biotin-binding agent. 
     
     
         47 . The method of  claim 44 , wherein the molecule of interest is an enzyme. 
     
     
         48 . The method of  claim 44 , wherein the molecule of interest is a polymerase. 
     
     
         49 . The method of  claim 44 , wherein the molecular complex of interest is a polymerase/template/primer complex. 
     
     
         50 . The method of  claim 43 , wherein the tetravalent biotin-binding agent is streptavidin. 
     
     
         51 . The method of  claim 38 , wherein L 3  is a linking group comprising a negatively charged group and L 4  is a linking group comprising a negatively charged group. 
     
     
         52 . The method of  claim 38 , wherein L 3  and L 4  comprise a sulfonic acid moiety. 
     
     
         53 . The method of  claim 38 , wherein L 3  and L 4  comprise a methylsulfonic acid group. 
     
     
         54 . The compound of  claim 32 , wherein L 3  and L 4  are identical groups selected from the group consisting of: