Protein nanopores with covalently attached molecular adapters

Molecular adapters are crucial for the stochastic sensing of organic analytes with (x-hemolysin (alpha HL) protein nanopores when direct interactions between analytes and the pore cannot readily be arranged by conventional protein engineering. In our earlier studies, cyclodextrin adapters were lodged noncovalently within the lumen of the (alpha HL pore. In the present work, we have realized the controlled covalent attachment of a beta-cycloclextrin (beta CD) adapter in the two possible molecular orientations inside alpha HL pores prepared by genetic engineering. There are two advantages to such a covalent system. First, the adapter cannot dissociate, which means there are no gaps during stochastic detection, a crucial advance for single-molecule exonuclease DNA sequencing where the continuous presence of a molecular adapter will be essential for reading individual nucleotides. Second, the ability to orient the adapter allows analytes to bind through only one of the two entrances to the PCD cavity. We demonstrate that the covalently attached adapters can be used to alter the ion selectivity of the alpha HL pore, examine binding events at elevated temperatures, and detect analytes with prolonged dwell times.