Pore-forming protein conjugate compositions and methods (trustees of columbia university in city of new york) gas yojana

Throughout this application, various publications and patents are referenced. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications and patents in their entirety are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. BACKGROUND

“Click” chemistry refers to a class of chemical reactions that are capable of quickly and efficiently forming covalent linkages between molecules. A variety of click chemical reactions are known, and many have been employed to conjugate biomolecules. In many cases, the click chemistry used to conjugate biomolecules is also “bio-orthogonal,” which means that reaction can occur in a biological system without interfering substantially with the normal biochemical function of that system. Such bio-orthogonal click reactions have been used extensively for in vivo labeling of biomolecules.

Single-molecule sequencing-by-synthesis (SBS) techniques using nanopores have been developed. See e.g., US Pat. Publ. Nos. 2013/0244340 A1 and 2013/0264207 A1. Nanopore SBS involves the use of a polymerase synthesize a DNA strand complementary to a target sequence template and determine the identity of each nucleotide monomer as it is added to the growing strand, thereby determining the target sequence. Each added nucleotide monomer is detected via a nanopore located adjacent to the polymerase active site and the growing strand. Obtaining an accurate signal requires proper positioning of a polymerase active site near a nanopore. Proper positioning typically is achieved by covalently linking the polymerase to the pore-protein that makes up the nanopore.

Monomeric pore-forming proteins have molecular weights range from as little as 5 kDa to 80 kDa, and these monomers form large multimeric complexes of 6, 7, 8, 3, 10, or more monomers, having molecular weights of 160, kDa, 180 kDa, 200 kDa, 220 kDa, or more. Under suitable conditions these multimeric complexes spontaneously form pores through lipid bilayer membranes. The well-studied pore-forming protein, α-hemolysin (from S. aureus) has a monomer molecular weight of 33 kDa and spontaneously forms a heptameric pore complex having a molecular weight of 231 kDa. Polymerases are large proteins that range in molecular weight from about 60 kDa to 100 kDa and even much larger multimeric complexes in some cases (e.g., RNA polymerase ˜400 kDa multimer). The Klenow fragment of DNA polymerase I has a molecular weight of 68 kDa.

Accordingly, the kinetics of any reaction to conjugate these pore-forming proteins, like the α-hemolysin heptamer, to large biomolecules, like DNA polymerase, in order to provide a nanopore sensor will be extremely limited by the low concentration achievable (and relative low amounts available) with such large macromolecules. The maximum solubility of such large proteins in aqueous solution typically is limited to approximately 0.1 to 10 mg/mL. Thus, the concentration of the two macromolecules in solution used for a conjugation reaction is limited to ˜1 μM to 1000 μM. For example, the α-hemolysin protein pore consists of 7 identical subunits totaling about 235,000 molecular weight. Thus a solution of 10 mg/ml has a concentration of about 42 μM. This relatively low concentration range effectively limits viable conjugation chemistries to those having extremely fast, irreversible reaction rates.

The typical Cu-catalyzed “click” (azide-alkyne Huisgen cycloaddition) chemistry is both slow and requires a copper (Cu) catalyst that can inactivate proteins and enzymes (See e.g., Wang et al. (2003) and Presolski et al. (2011)). More recently, copper-free cycloaddition involving alkynes and asides have been developed (See e.g., Jewett and Bertozzi (2010)). These Cu-free reactions, however, are too slow for practical conjugation reactions between two large protein molecules, and can require 3-4 days to provide a significant yield of conjugates. Many proteins of interest, particularly enzymes such as polymerases, cannot withstand such long reaction times.

Due to the relatively low-concentrations of pore-protein and polymerase typically used in forming a nanopore detector for SBS, it is important that a highly efficient and selective chemical “click” reaction is developed allowing strong, selective, covalent conjugation between the two. Thus, there remains a need for faster and more efficient chemical processes to manufacture the polymerase-pore structures used in nanopore sequencing. SUMMARY

The present disclosure provides compositions of a conjugate between a pore-forming protein and a biomolecule, and chemical processes for preparing such pore-forming protein conjugates, and their incorporation in nanopores and associated uses, including use in nanopore sequencing.

wherein, one of A and B is a pore-forming protein, and the other a biomolecule selected from the group consisting of an enzyme, an oligonucleotide (e.g., of at least about 20 nucleotides), an antibody, and a receptor; LA and LB are linkers; X1 and X2 are atoms independently selected from C and N; and R1 is a chemical group selected from the group consisting of: H, F, Cl, Br, I, CH3, CF3, NH2, NO2, OH C(O)OH, C(O)OCH3, C(O)NH2, linear or branched (C2-C5) alkyl, linear or branched (C2-C5) alkenyl, linear or branched (C2-C5) alkynyl, unsubstituted or para-substituted 6-membered aryl ring, and unsubstituted or para-substituted 6-membered heteroaryl ring. In some embodiments, the composition further comprises an aqueous solution.

In some embodiments, the present disclosure provides a nanopore composition comprising a compound of formula (I), wherein one of A and B is a pore-forming protein is part of a nanopore. In same embodiments, the nanopore is embedded in a membrane, and optionally, the membrane can be attached to a solid substrate, and/or is formed such that it spans a well or depression or hole in a solid substrate, which optionally comprises a material selected from the group consisting of polymer, glass, silicon, and a combination thereof. In some embodiments, the solid substrate further comprises adjacent to the nanopore 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.

In some embodiments, the present disclosure provides a method of preparing a composition comprising a compound of formula (I), wherein the method comprises contacting under suitable reaction conditions: (a) a tetrazine-Iinker-conjugate compound of formula (II)

wherein, one of A and B is a pore-forming protein, and the other is a biomolecule capable of catalyzing the synthesis of a nucleotide polymer; LA and LB are linkers; X1 and X2 are atoms independently selected from C and N; and R1 is a chemical group selected from the group consisting of: H, F, Cl, Br, I, CH3, CF3, NH2, NO2, OH, C(O)OH, C(O)OCH3, C(O)NH2, linear or branched (C2-C5) alkyl, linear or branched (C2-C5) alkenyl, linear or branched (C2-C5) alkynyl, unsubstituted or para-substituted 6-membered aryl ring, and unsubstituted or para-substituted 6-membered heteroaryl ring; and whereby the contacted compounds of formula (II) and formula (III) undergo an inverse electron demand Diels-Alder (IEDDA) reaction to form a conjugate of the pore-forming protein and the biomolecule.

In some embodiments, the present disclosure provides 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: