Single molecule nanopore sequencing method

A construct comprising a transmembrane protein pore subunit and a polynucleotide-binding protein, wherein the subunit retains its pore-forming ability, the polynucleotide-binding protein is capable of unwinding the two strands of a double-stranded polynucleotide and/or controlling the movement of a single-stranded polynucleotide through the pore;

• and, the subunit is in covalent binding to the polynucleotide-binding protein;
• preferably, the covalent bonding is not via a peptide bond generated by nucleic acid translation;
• preferably, the covalent binding is via an isopeptide bond;
• preferably, the covalent binding occurs through chemical ligation (e.g., by click chemistry).