Pore-forming protein conjugate compositions and methods

Abstract

The present disclosure relates to compositions and methods based on a fast, efficient chemical reaction for conjugating a pore-forming protein, such as α-hemolysin, to a biomolecule, such as antibodies, receptors, and enzymes, such as DNA polymerase, and the use of such pore-forming conjugates in nanopore devices and methods.

What is claimed is: 
     
         1 . A composition comprising a compound of formula (I), 
       
         
           
           
               
               
           
         
         wherein
 one of A and B is a pore-forming protein, and the other is a biomolecule selected from the group consisting of an enzyme, an oligonucleotide of at least 20 nucleotides, an antibody, and a receptor; 
 L A  and L B  are linkers; 
 X 1  and X 2  are atoms independently selected from C and N; and 
 R 1  is a chemical group selected from the group consisting of: H, F, Cl, Br, I, CH 3 , CF 3 , NH 2 , NO 2 , OH, C(O)OH, C(O)OCH 3 , C(O)NH 2 , linear or branched (C 2 -C 5 ) alkyl, linear or branched (C 2 -C 5 ) alkenyl, linear or branched (C 2 -C 5 ) alkynyl, unsubstituted or para-substituted 6-membered aryl ring, and unsubstituted or para-substituted 6-membered heteroaryl ring. 
 
       
     
     
         2 . The composition of  claim 1 , wherein the pore-forming protein has a molecular weight of at least 30 kDa and the biomolecule has a molecular weight of at least 50 kDa. 
     
     
         3 . The composition of any one of  claims 1 - 2 , wherein the pore-forming protein:
 a) is a part of a multimeric complex, including a heptamer;   b) is part of a nanopore   c) is capable of forming a nanopore of a diameter of about 0.5 nanometer to about 25 nanometers;   d) is embedded in a membrane;   e) is attached to a solid substrate, including substrates comprising a material selected from the group consisting of polymer, glass, silicon, and a combination thereof; and/or   f) is selected from the group consisting of α-hemolysin, α-HL-C46, β-hemolysin, γ-hemolysin, aerolysin, cytolysin, leukocidin, melittin, MspA porin and porin A.   
     
     
         4 . The composition of any one of  claims 1 - 3 , wherein the composition is in aqueous solution. 
     
     
         5 . The composition of any one of  claims 1 - 4 , wherein the biomolecule:
 a) is an enzyme capable of catalyzing the synthesis of a polymer;   b) is an enzyme selected from the group consisting of a DNA polymerase, RNA polymerase, reverse transcriptase, terminal transferase, helicase, and DNA ligase;   c) comprises a large fragment of DNA polymerase from  B. stearothermophilus;      d) is DNA polymerase Bst 2.0; and/or   e) 9°N polymerase,  E. Coli  DNA Polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, Tag DNA polymerase, 9°N polymerase (exo-A)A485L/Y409V or Phi29 DNA polymerase (ϕ29 DNA Polymerase).   
     
     
         6 . The composition of any one of  claims 1 - 5 , wherein the X 1  and X 2  atoms are each C, and R 1  is a CH 3  group. 
     
     
         7 . The composition of any one of  claims 1 - 5 , wherein one or both of the X 1  and X 2  atoms are N. 
     
     
         8 . The composition of any one of  claims 1 - 5 , wherein the R 1  is an unsubstituted or para-substituted 6-membered aryl ring, or an unsubstituted or para-substituted 6-membered heteroaryl ring selected from the group consisting of phenyl, benzoic acid, 4-methyl-phenyl, 4-methoxy-phenyl, 4-trifluoromethyl-phenyl, 2-pyridyl, 2-pyridyl-4-methyl, 2-pyridyl-4-carboxylic acid, 2-pyrimidyl, 2-pyrimidyl-4 -methyl, and 2-pyrimidyl-4-carboxylic acid. 
     
     
         9 . The composition of any one of  claims 1 - 5 , wherein the compound of formula (I) is selected from the group consisting of compounds of formula (Ia) to formula (Iq): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         10 . The composition of any one of  claims 1 - 9 , wherein the linkers L A  and L B  comprise a covalently bonded chain of 2 to 100 atoms comprising one or more of the following chemical groups: linear (C 1 -C 5 ) alkyl, linear (C 1 -C 5 ) alkenyl, linear (C 1 -C 5 ) alkynyl, ester, ether, amine, amide, imide, phosphodiester, and/or polyethylene glycol (PEG). 
     
     
         11 . The composition of any one of  claims 1 - 10 , wherein the linkers L A  and L B  attach to A and B either through a thioether bond to a sulfhydryl group on A and/or B, or through a peptide bond to a primary amine group of A and/or B. 
     
     
         12 . The composition of  claim 11 , wherein the linkers L A  and L B  are attached to A and B, respectively, through a thioether bond to a sulfhydryl group on A and B. 
     
     
         13 . The composition of any one of  claims 10 - 12 , wherein the linkers L A  and L B  comprise a polymer of from 1 to 50 polyethylene glycol (PEG) moieties. 
     
     
         14 . The composition of any one of  claims 1 - 13 , wherein the linkers L A  and L B  are independently selected from the group consisting of structures of formula (IVa)-formula (IVd): 
       
         
           
           
               
               
           
         
       
     
     
         15 . The composition of any one of  claims 1 - 13 , wherein the compound of formula (I) are independently selected from the group consisting of structures of formula (Ir), (Is), or (It): 
       
         
           
           
               
               
           
         
         wherein, n=1 to 50, and q, r, and s each independently=0, 1, 2 or 3 
       
       
         
           
           
               
               
           
         
         wherein, n=1 to 50, and q, r, and s each independently=0, 1, 2, or 3; 
       
       
         
           
           
               
               
           
         
       
     
     
         16 . The composition of any one of  claims 1 - 15 , wherein the composition is a nanopore composition. 
     
     
         17 . The composition  claim 16 , wherein the nanopore comprises a 6:1 ratio of native α-HL monomer to α-HL-C46 monomer. 
     
     
         18 . The composition of any one of  claims 16 - 17 , wherein the nanopore is embedded in a membrane, wherein the membrane is attached to a solid substrate comprising a material selected from the group consisting of polymer, glass, silicon, and a combination thereof, and wherein the solid substrate comprises a sensor, a sensing circuit, or an electrode coupled to a sensing circuit, optionally, a complementary metal-oxide semiconductor (CMOS), or field effect transistor (FET) circuit. 
     
     
         19 . A method of preparing a conjugate of a pore-forming protein and a biomolecule capable of catalyzing the synthesis of a nucleotide polymer, said method comprising contacting under suitable reaction conditions:
 a tetrazine-linker-conjugate compound of formula (II)   
       
         
           
           
               
               
           
         
       
       and 
       a TCO-linker-conjugate compound of formula (III), 
       
         
           
           
               
               
           
         
         wherein,
 one of A and B is a pore-forming protein, and the other a biomolecule capable of catalyzing the synthesis of a nucleotide polymer; 
 L A  and L B  are linkers; 
 X 1  and X 2  are atoms independently selected from C and N; and 
 R 1  is a chemical group selected from the group consisting of: H, F, Cl, Br, I, CH 3 , CF 3 , NH 2 , NO 2 , OH, C(O)OH, C(O)OCH 3 , C(O)NH 2 , linear or branched (C 2 -C 5 ) alkyl, linear or branched (C 2 -C 5 ) alkenyl, linear or branched (C 2 -C 5 ) alkynyl, unsubstituted or peril-substituted 6-membered aryl ring, and unsubstituted or para-substituted 6-membered heteroaryl ring; 
 whereby the compounds undergo an inverse electron demand Diels-Alder reaction to form a conjugate of the pore-forming protein and the biomolecule. 
 
       
     
     
         20 . The method of  19 , wherein the R 1  is an unsubstituted or para-substituted 6-membered aryl ring, or an unsubstituted or para-substituted 6-membered heteroaryl ring selected, from the group consisting of: phenyl, benzoic acid, 4-methyl-phenyl, 4-methoxy-phenyl, 4-trifluoromethyl-phenyl, 2-pyridyl, 2-pyridyl-4-methyl, 2-pyridyl -4-carboxylic acid, 2-pyrimidyl, 2-pyrimidyl-4-methyl, and 2-pyrimidyl-4-carboxylic acid. 
     
     
         21 . The method of any one of  claims 19 - 20 , wherein the tetrazine-linker-conjugate compound of formula (II) is a compound of formula (IIo) or (IIp): 
       
         
           
           
               
               
           
         
         wherein, n=1 to 50,and q, r, and s each independently=0, 1, 2, or 3; 
       
       
         
           
           
               
               
           
         
       
     
     
         22 . The method of any one of  claims 19 - 20 , wherein the TCO-linker-conjugate compound of formula (III) is a compound of formula (IIId) or (IIIe): 
       
         
           
           
               
               
           
         
         wherein, n=1 to 50, and q, r, and s each independently=0, 1, 2, or 3; 
       
       
         
           
           
               
               
           
         
       
     
     
         23 . A method of preparing a conjugate of a pore-forming protein and a biomolecule capable of catalyzing the synthesis of a nucleotide polymer, said method comprising the steps of:
 (a) contacting a pore-forming protein with a tetrasine-linker-maleimide compound of formula (V) under suitable reaction conditions:   
       
         
           
           
               
               
           
         
       
       wherein,
 X 1  and X 2  are atoms independently selected from C and N; 
 R 1  is a chemical group selected from the group consisting of: H, F, Cl, Br, I, CH 3 , CF 3 , NH 2 , NO 2 , OH C(O)OH, C(O)OCH 3 , C(O)NH 2 , linear or branched (C 2 -C 5 ) alkyl, linear or branched (C 2 -C 5 ) alkenyl, linear or branched (C 2 -C 5 ) alkynyl, unsubstituted or para-substituted 6-membered aryl ring, and unsubstituted or para-substituted 6-membered heteroaryl ring; and 
 n=1 to 50, and q, r, and s each independently =0, 1, 2, or 3; 
 
       thereby forming a tetrazine-linker-pore-forming protein conjugate;
 (b) contacting a biomolecule capable of catalyzing the synthesis of a nucleotide polymer with a trans-cyclooctene-linker-maleimide compound of formula (VI) under suitable reaction conditions, 
 
       
         
           
           
               
               
           
         
       
       wherein, n=1 to 50, and q, r, and s each independently=0, 1, 2, or 3;
 thereby forming a TCO-linker-biomolecule conjugate; and 
 (c) contacting the tetrazine-linker-pore-forming protein conjugate of step (a) with the TCO-linker-biomolecule conjugate of step (b) under suitable reaction conditions,
 whereby the two conjugates undergo an inverse electron demand Diels-Alder reaction to form a conjugate of the pore-forming protein and the biomolecule. 
 
 
     
     
         24 . The method of  claim 23 , wherein the tetrazine-linker-maleimide compound is a compound of formula (Va): 
       
         
           
           
               
               
           
         
         wherein, n=1 to 24, and q, r, and s each independently=1, 2 or 3. 
       
     
     
         25 . The method of any one of  claims 23  or  24 , wherein the tetrazine-linker-maleimide compound is a compound of formula (Va): 
       
         
           
           
               
               
           
         
         wherein, n=1 to 24, and q, r, and s each independently=1, 2 or 3. 
       
     
     
         26 . The method of any one of  claims 23 - 25 , wherein the TCO-linker-maleimide compound is compound (6): 
       
         
           
           
               
               
           
         
       
     
     
         27 . The method of any one of  claims 19 - 26 , wherein the pore-forming protein has a molecular weight of at least 30 kDa and the biomolecule has a molecular weight of at least 50 kDa. 
     
     
         28 . The method of any one of  claims 19 - 27 , wherein the pore-forming protein:
 a) is a part of a multimeric complex;   b) is selected from the group consisting of α-hemolysin, α-L-C46, β-hemolysin, γ-hemolysin, aerolysin, cytolysin, leukocidin, melittin, MspA porin and porin;   c) is capable of forming a nanopore of diameter of about 0.5 nanometer to about 25 nanometers;   d) is embedded in a membrane;   e) is part of a nanopore;   f) protein is attached to a solid substrate; and/or   g) is attached to a solid substrate comprising a material selected from the group consisting of polymer, glass, silicon, and a combination thereof.   
     
     
         29 . The method of any one of  claims 19 - 28 , wherein the biomolecule is:
 a) an enzyme capable of catalyzing the synthesis of a polymer;   b) an enzyme selected from the group consisting of a DNA polymerase, RNA polymerase, reverse transcriptase, terminal transferase, helicase and DNA ligase;   c) comprises the large fragment of DNA polymerase from  S. stearothermophilus;      d) DNA polymerase Bst 2.0; and/or   e) 9° N polymerase,  E. Coli  DNA Polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, Tag DNA polymerase, 9°N polymerase (exo-)A485L/Y409V or Phi29 DNA polymerase (ϕ29 DNA Polymerase).