Single-molecule fingerprinting of protein-drug interaction using a funneled biological nanopore

Abstract

In drug discovery, efficient screening of protein-drug interactions (PDIs) is hampered by the limitations of current biophysical approaches. Here, we develop a biological nanopore sensor for single-molecule detection of proteins and PDIs using the pore-forming toxin YaxAB. Using this YaxAB nanopore, we demonstrate label-free, single-molecule detection of interactions between the anticancer Bcl-xL protein and small-molecule drugs as well as the Bak-BH3 peptide. The long funnel-shaped structure and nanofluidic characteristics of the YaxAB nanopore enable the electro-osmotic trapping of diverse folded proteins and high-resolution monitoring of PDIs. Distinctive nanopore event distributions observed in the two-dimensional (ΔI/Io-versus-IN) plot illustrate the ability of the YaxAB nanopore to discriminate individual small-molecule drugs bound to Bcl-xL from non-binders. Taken together, our results present the YaxAB nanopore as a robust platform for label-free, ultrasensitive, single-molecule detection of PDIs, opening up a possibility for low-cost, highly efficient drug discovery against diverse drug targets.

Protein Sizing with 15 nm Conical Biological Nanopore YaxAB

“…Most likely, this effect is due to deeper penetration of smaller proteins in the transmembrane region. Proteins smaller than the constriction region would translocate, and extended sampling of smaller proteins would require smaller YaxAB pores, as shown in recent work published during the peer review of this work …”

“…Proteins smaller than the constriction region would translocate, and extended sampling of smaller proteins would require smaller YaxAB pores, as shown in recent work published during the peer review of this work. 59 YaxAB might be particularly useful to characterize large proteins that typically cannot be addressed by other nanopores. We tested CRP (125 kDa), an important inflammatory biomarker whose concentration in blood is related to sepsis, which is one of the main causes of death in patients in intensive care units.…”

Lipid vesicle-based molecular robots

“…67 Following the achievement of nanopore DNA sequencing (Fig. 4(a)), single-molecule analysis of folded proteins [68][69][70][71][72] and amino acid sequences in proteins [73][74][75] are now underway. Advances such as the detection of single amino acid mutations in peptides using aerolysin from Aeromonas hydrophila, 76 sequential readout of peptides using MspA, 77,78 and identification of digested protein fragments using fragaceatoxin C from Actinia fragacea (FraC) 79 have been reported in recent years.…”

Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer Applications

“…A number of emerging sensing technologies critically depend on robust lipid bilayers, including those based on biological nanopores. − To work properly, analytes must be able to move from the bathing solution surrounding the interface to the mouth of the nanopore. Specific nanopore sensing applications include DNA sequencing, RNA sequencing, − nucleic acid detection, − polypeptide detection, RNA profiling, synthetic polymer characterization, − digital data storage, − disease detection, ion sensing, small molecule detection, and sensing of protein–drug interactions . Multiple types of nanopores with various pore sizes and channel structures can be employed.…”