Pore-forming protein conjugate compositions and methods

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 ,

i) 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, and/or

ii) 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) is 9° N polymerase, E. Coli DNA Polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, Taq DNA polymerase, 9° N polymerase (exo-)A485L/Y409V or Phi29 DNA polymerase (φ29 DNA Polymerase), and/or

iii) 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 claim 1 ,

a) wherein the X 1 and X 2 atoms are each C, and R 1 is a CH 3 group,

b) wherein one or both of the X 1 and X 2 atoms are N, or

c) 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.

4. The composition of claim 1 , wherein the compound of formula (I) is selected from the group consisting of compounds of formula (Ia) to formula (Iq):

5. The composition of claim 1 ,

a) 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), and/or

b) 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, and optionally 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, and

c) wherein optionally the linkers L A and L B comprise a polymer from 1 to 50 polyethylene glycol (PEG) moieties.

6. The composition of claim 1 , wherein the linkers L A and L B are independently selected from the group consisting of structures of formula (IVa)-formula (IVd):

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;

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.

7. The composition of claim 1 , 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;

8. The composition of claim 1 , wherein the composition is a nanopore composition.

9. The composition of claim 8 , wherein the nanopore comprises a 6:1 ratio of native α-HL monomer to α-HL-C46 monomer, and optionally 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.

10. 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 para-substituted 6-membered aryl ring, and unsubstituted or para-substituted 6-membered heteroaryl ring, including those 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;

whereby the compounds undergo an inverse electron demand Diels-Alder reaction to form a conjugate of the pore-forming protein and the biomolecule.

11. The method of claim 10 , 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;

12. The method of claim 10 , 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;

13. 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 tetrazine-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.

14. The method of claim 13 , 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.

15. The method of claim 13 , 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.

16. The method of claim 13 , wherein the TCO-linker-maleimide compound is compound (6):

17. The method of claim 13 , 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.

18. The method of claim 13 ,

i) wherein the pore-forming protein:

a) is a part of a multimeric complex;

b) is selected from the group consisting of α-hemolysin, α-HL-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, and/or

ii) 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 B. stearothermophilus;

d) DNA polymerase Bst 2.0; and/or

e) 9° N polymerase, E. Coli DNA Polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, Taq DNA polymerase, 9° N polymerase (exo-)A485L/Y409V or Phi29 DNA polymerase (φ29 DNA Polymerase).