Target sequences for synthetic molecules

Abstract

The invention is based on the discovery that certain biarsenical molecules react with specified target sequences, thereby providing a facile means for labeling polypeptides containing the target sequence. The invention is useful in creating stable mammalian cell lines expressing a certain tetracysteine tagged polypeptides, thereby overcoming toxicity associated with native tetracysteine. In addition, the invention allows for orthogonal labeling of polypeptides, thereby allowing for the observation of protein-protein interactions and conformational changes in proteins, for example.

1 . A vector comprising a nucleic acid sequence encoding a bonding partner, wherein the nucleic acid sequence encoding the bonding partner comprises a nucleic acid sequence encoding a carrier polypeptide and a nucleic acid sequence encoding a target sequence, wherein the nucleic acid sequence encoding the target sequence is heterologous to the nucleic acid sequence encoding the carrier polypeptide, and the target sequence comprises at least 2 cysteines and has the sequence of Cys-Cys-R n , whether R is any amino acid and n is an integer from 1-100, and wherein when n≧2, each R is selected independently from every other R in the sequence, but wherein the target sequence cannot be Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ. ID NO: 1) wherein Xaa can be any amino acid, and wherein the target sequence reacts with a biarsenical molecule having the formula:  
       
         
           
           
               
               
           
         
       
       and tautomers, anhydrides, and salts thereof; 
 wherein: 
 each X 1  or X 2  is independently Cl, Br, I, OR a , or SR a , or  
 X 1  and X 2  together with the arsenic atom form a ring having the formula:  
                     
 R a  is H, C 1 -C 4  alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;  
 Z is 1,2-ethanediyl, 1,2-propanediyl, 2,3-butanediyl, 1,3-propanediyl, 1,2 benzenediyl, 4-methyl-1,2-benzenediyl, 1,2-cyclopentanediyl, 1,2-cyclohexanediyl, 3-hydroxy-1,2-propanediyl, 3-sulfo-1,2-propanediyl, or 1,2-bis(carboxy)-1,2-ethanediyl;  
 Y 1  and Y 2 , are each independently H or CH 3 , or  
 Y 1  and Y 2  together form a ring such that the biarsenical molecule has the formula  
                     
 
 wherein: 
 M is O, S, CH 2 , C(CH 3 ) 2 , or NH;  
 R 1  and R 2  are each independently OR a , OAc, NR a R b , or H;  
 R 3  and R 4  are each independently H, F, Cl, Br, I, OR a , or R a ; or  
 R 1  together with R 3 , or R 2  together with R 4 , or both, form a ring in which  
 
 (i) one of R 1  or R 3  is C 2 -C 3  alkyl and the other is NR a  and  
 (ii) one of R 2  and R 4  is C 2 -C 3  alkyl and the other is NR a ; 
 R b  is H, C 1 -C 4  alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;  
 Q is CR a R b , CR a OR b , C═O, or a spirolactone having the formula:  
                     
 
 wherein the spiro linkage is formed at C 1    
 
     
     
         2 . The vector of  claim 1 , wherein the target sequence is Cys-Cys-X 1 -X 1 -X 2 -X 1 -X 3 -X 1 -X 1 -Cys-X 1 -Cys-X 2  (SEQ. ID NO: 3), wherein: 
 X 1  is an amino acid having a non-polar side chain,    X 2  is an amino acid having a basic side chain, and    X 3  is an amino acid having a non-ionic polar side chain.    
     
     
         3 . The vector of  claim 2 , wherein X 1  is glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan.  
     
     
         4 . The vector of  claim 2 , wherein X 2  is lysine, arginine, or histidine.  
     
     
         5 . The vector of  claim 2 , wherein X 3  is asparagine, glutamine, serine, or threonine.  
     
     
         6 . The vector of  claim 1 , wherein the target sequence is Cys-Cys-gly-gly-lys-gly-asn-gly-gly-Cys-gly-Cys-his (SEQ. ID NO: 50).  
     
     
         7 . The vector of  claim 1 , wherein the nucleic acid sequence encoding the target sequence is attached at the 5′-end of the nucleic acid sequence encoding the carrier polypeptide.  
     
     
         8 . The vector of  claim 1 , wherein the nucleic acid sequence encoding the target sequence is attached at the 3′-end of the nucleic acid sequence encoding the carrier polypeptide.  
     
     
         9 . The vector of  claim 1 , wherein the biarsenical molecule has the following structure:  
       
         
           
           
               
               
           
         
       
     
     
         10 . A method of labeling a carrier molecule, comprising: 
 a) providing a bonding partner comprising the carrier molecule and a target sequence, and    b) contacting the bonding partner with a biarsenical molecule under conditions wherein the biarsenical molecule reacts with the target sequence, wherein the target sequence comprises at least 2 cysteines and has the sequence Cys-Cys-R n , wherein R is any amino acid and n is an integer from 1-100, and wherein when n≧2, each R is selected independently from every other R in the sequence, but wherein the target sequence cannot be Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ. ID NO: 1) wherein Xaa can be any amino acid, and wherein the biarsenical molecule has the formula:                          and tautomers, anhydrides, and salts thereof;    wherein: 
 each X 1  or X 2  is independently Cl, Br, I, OR a , or SR a , or  
 X 1  and X 2  together with the arsenic atom form a ring having the formula:  
                     
 R a  is H, C 1 -C 4  alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;  
 Z is 1,2-ethanediyl, 1,2-propanediyl, 2,3-butanediyl, 1,3-propanediyl, 1,2 benzenediyl, 4-methyl-1,2-benzenediyl, 1,2-cyclopentanediyl, 1,2-cyclohexanediyl, 3-hydroxy-1,2-propanediyl, 3-sulfo-1,2-propanediyl, or 1,2-bis(carboxy)-1,2-ethanediyl;  
 Y 1  and Y 2  are each independently H or CH 3 , or  
 Y 1  and Y 2  together form a ring such that the biarsenical molecule has the formula  
                     
   wherein: 
 M is O, S, CH 2 , C(CH 3 ) 2 , or NH;  
 R 1  and R 2  are each independently OR a , OAc, NR a R b , or H;  
 R 3  and R 4  are each independently H, F, Cl, Br, I, OR a , or R a ; or  
 R 1  together with R 3 , or R 2  together with R 4 , or both, form a ring in which  
   (i) one of R 1  or R 3  is C 2 -C 3  alkyl and the other is NR a  and    (ii) one of R 2  and R 4  is C 2 -C 3  alkyl and the other is NR a ; 
 R b  is H, C 1 -C 4  alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;  
 Q is CR a R b , CR a OR b , C═O, or a spirolactone having the formula:  
                     
   wherein the spiro linkage is formed at C 1 .    
     
     
         11 . The method of  claim 10 , wherein the biarsenical molecule generates a detectable signal.  
     
     
         12 . The method of  claim 11 , further comprising monitoring the detectable signal.  
     
     
         13 . The method of  claim 11 , wherein the signal is a fluorescent signal.  
     
     
         14 . The method of  claim 10 , wherein the biarsenical molecule is coupled to a solid phase.  
     
     
         15 . The method of  claim 10 , wherein the target sequence is coupled to a solid phase.  
     
     
         16 . The method of  claim 10 , wherein the carrier molecule is a polypeptide.  
     
     
         17 . The method of  claim 16 , wherein the polypeptide is an antibody or an enzyme.  
     
     
         18 . The method of  claim 10 , wherein the target sequence is Cys-Cys-X 1 -X 1 -X 2 -X 1 -X 3 -X 1 -X 1 -Cys-X 1 -Cys-X 2  (SEQ. ID NO: 3), wherein: 
 X 1  is an amino acid having a non-polar side chain,    X 2  is an amino acid having a basic side chain, and    X 3  is an amino acid having a non-ionic polar side chain.    
     
     
         19 . The method of  claim 18 , wherein X 1  is glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan.  
     
     
         20 . The method of  claim 18 , wherein X 2  is lysine, arginine, or histidine.  
     
     
         21 . The method of  claim 18 , wherein X 3  is asparagine, glutamine, serine, or threonine.  
     
     
         22 . The method of  claim 10 , wherein the target sequence is Cys-Cys-gly-gly-lys-gly-asn-gly-gly-Cys-gly-Cys-his (SEQ. ID NO: 50).  
     
     
         23 . A kit comprising: 
 a) a biarsenical molecule having the structure:                          and tautomers, anhydrides, and salts thereof;    wherein: 
 each X 1  or X 2  is independently Cl, Br, I, OR a , or SR a , or  
 X 1  and X 2  together with the arsenic atom form a ring having the formula:  
                     
 R a  is H, C 1 -C 4  alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;  
 Z is 1,2-ethanediyl, 1,2-propanediyl, 2,3-butanediyl, 1,3-propanediyl, 1,2 benzenediyl, 4-methyl-1,2-benzenediyl, 1,2-cyclopentanediyl, 1,2-cyclohexanediyl, 3-hydroxy-1,2-propanediyl, 3-sulfo-1,2-propanediyl, or 1,2-bis(carboxy)-1,2-ethanediyl;  
 Y 1  and Y 2  are each independently H or CH 3 , or  
 Y 1  and Y 2  together form a ring such that the biarsenical molecule has the formula  
                     
   wherein: 
 M is O, S, CH 2 , C(CH 3 ) 2 , or NH;  
 R 1  and R 2  are each independently OR a , OAc, NR a R b , or H;  
 R 3  and R 4  are each independently H, F, Cl, Br, I, OR a , or R a ; or  
 R 1  together with R 3 , or R 2  together with R 4 , or both, form a ring in which  
   (i) one of R 1  or R 3  is C 2 -C 3  alkyl and the other is NR a  and    (ii) one of R 2  and R 4  is C 2 -C 3  alkyl and the other is NR a ; 
 R b  is H, C 1 -C 4  alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;  
 Q is CR a R b , CR a OR b , C═O, or a spirolactone having the formula:  
                     
   wherein the spiro linkage is formed at C 1 ; and    b) a bonding partner comprising a target sequence, wherein the target sequence comprises at least 2 cysteines and has the sequence Cys-Cys-R n , wherein R is any amino acid and n is an integer from 1-100, and wherein when n≧2, each R is selected independently from every other R in the sequence, but wherein the target sequence cannot be Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ. ID NO: 1) wherein Xaa can be any amino acid, and wherein the target sequence reacts with the biarsenical molecule.    
     
     
         24 . The kit of  claim 23 , wherein the target sequence is Cys-Cys-X 1 -X 1 -X 2 -X 1 -X 3 -X 1 -X 1 -Cys-X 1 -Cys-X 2  (SEQ. ID NO: 3), wherein: 
 X 1  is an amino acid having a non-polar side chain,    X 2  is an amino acid having a basic side chain, and    X 3  is an amino acid having a non-ionic polar side chain.    
     
     
         25 . The kit of  claim 24 , wherein X 1  is glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan.  
     
     
         26 . The kit of  claim 24 , wherein X 2  is lysine, arginine, or histidine.  
     
     
         27 . The kit of  claim 24 , wherein X 3  is asparagine, glutamine, serine, or threonine.  
     
     
         28 . The kit of  claim 23 , wherein the target sequence is Cys-Cys-gly-gly-lys-gly-asn-gly-gly-Cys-gly-Cys-his (SEQ. ID NO: 50).  
     
     
         29 . A method of purifying a bonding partner, comprising: 
 a) providing a bonding partner comprising a target sequence, comprises at least 2 cysteines and has the sequence of Cys-Cys-R n , whether R is any amino acid and n is an integer from 1-100, and wherein when n≧2, each R is selected independently from every other R in the sequence, but wherein the target sequence cannot be Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ. ID NO: 1) wherein Xaa can be any amino acid, and wherein the target sequence reacts with a biarsenical molecule having the structure:                          and tautomers, anhydrides, and salts thereof;    wherein: 
 each X 1  or X 2  is independently Cl, Br, I, OR a , or SR a , or  
 X 1  and X 2  together with the arsenic atom form a ring having the formula:  
                     
 R a  is H, C 1 -C 4  alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;  
 Z is 1,2-ethanediyl, 1,2-propanediyl, 2,3-butanediyl, 1,3-propanediyl, 1,2 benzenediyl, 4-methyl-1,2-benzenediyl, 1,2-cyclopentanediyl, 1,2-cyclohexanediyl, 3-hydroxy-1,2-propanediyl, 3-sulfo-1,2-propanediyl, or 1,2-bis(carboxy)-1,2-ethanediyl;  
 Y 1  and Y 2  are each independently H or CH 3 , or  
 Y 1  and Y 2  together form a ring such that the biarsenical molecule has the formula  
                     
   wherein: 
 M is O, S, CH 2 , C(CH 3 ) 2 , or NH;  
 R 1  and R 2  are each independently OR a , OAc, NR a R b , or H;  
 R 3  and R 4  are each independently H, F, Cl, Br, I, OR a , or R a ; or  
 R 1  together with R 3 , or R 2  together with R 4 , or both, form a ring in which  
   (i) one of R 1  or R 3  is C 2 -C 3  alkyl and the other is NR a  and    (ii) one of R 2  and R 4  is C 2 -C 3  alkyl and the other is NR a ; 
 R b  is H, C 1 -C 4  alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;  
 Q is CR a R b , CR a OR b , C═O, or a spirolactone having the formula:  
                     
   wherein the spiro linkage is formed at C 1 ;    d) contacting the bonding partner with the biarsenical molecule, wherein the biarsenical molecule is coupled to a solid phase,    e) eluting the bonding partner from the biarsenical molecule by contacting the biarsenical molecule with a dithiol.    
     
     
         30 . The method of  claim 29 , wherein the carrier molecule is a protein.  
     
     
         31 . The method of  claim 30 , wherein the protein is an antibody or an enzyme.  
     
     
         32 . The method of  claim 29 , wherein the biarsenical compound is membrane permeable.  
     
     
         33 . The method of  claim 29 , wherein the dithiol is selected from the group consisting of 1,2-benzendithiol, 1,2-cyclohexanedithiol and 1,2-ethanedithiol.  
     
     
         34 . An isolated polypeptide comprising a sequence Cys-Cys-X 1 -X 1 -X 2 -X 1 -X 3 -X 1 -X 1 -Cys-X 1 -Cys-X 2  (SEQ. ID NO: 3), wherein: 
 X 1  is an amino acid having a non-polar side chain,    X 2  is an amino acid having a basic side chain, and    X 3  is an amino acid having a non-ionic polar side chain.    
     
     
         35 . The polypeptide of  claim 34 , wherein X 1  is glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan.  
     
     
         36 . The polypeptide of  claim 34 , wherein X 2  is lysine, arginine, or histidine.  
     
     
         37 . The polypeptide of  claim 34 , wherein X 3  is asparagine, glutamine, serine, or threonine.  
     
     
         38 . An isolated polypeptide comprising a sequence Cys-Cys-Gly-Gly-Lys-Gly-Asn-Gly-Gly-Cys-Gly-Cys-His (SEQ. ID. NO: 50).  
     
     
         39 . An isolated nucleic acid molecule encoding a polypeptide having a sequence Cys-Cys-X 1 -X 1 -X 2 -X 1 -X 3 -X 1 -X 1 -Cys-X 1 -Cys-X 2  (SEQ. ID NO: 3), wherein: 
 X 1  is an amino acid having a non-polar side chain,    X 2  is an amino acid having a basic side chain, and    X 3  is an amino acid having a non-ionic polar side chain.    
     
     
         40 . The isolated nucleic acid molecule of  claim 39 , wherein X 1  is glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan.  
     
     
         41 . The isolated nucleic acid molecule of  claim 39 , wherein X 2  is lysine, arginine, or histidine.  
     
     
         42 . The isolated nucleic acid molecule of  claim 39 , wherein X 3  is asparagine, glutamine, serine, or threonine.  
     
     
         43 . An isolated nucleic acid molecule encoding a polypeptide having a sequence Cys-Cys-Gly-Gly-Lys-Gly-Asn-Gly-Gly-Cys-Gly-Cys-His (SEQ. ID. NO: 50).