Pore-forming protein conjugate compositions and methods

Patent metadata

Field	Value
Publication number	US-9890426-B2
Application number	US-201715439173-A
Country	US
Kind code	B2
Filing date	Feb 22, 2017
Priority date	Mar 9, 2015
Publication date	Feb 13, 2018
Grant date	Feb 13, 2018

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 protein conjugates in nanopore devices and methods.

First claim

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 ,
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

Assignees

• Ju Jingyue
• Li Zengmin
• Kalachikov Sergey
• Fuller Carl
• Univ Columbia

Inventors

• Ju Jingyue
• Li Zengmin
• Kalachikov Sergey
• Fuller Carl

Classifications

• A61K47/69

  the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit · CPC title

• C12N9/1252

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C07K19/00

  Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title