Oral Presentation The 6th Prato Conference on Pore Forming Proteins 2025

Stochastic Sensing of Chloride Anions Using an α-Hemolysin Pore with a semiaza-Bambusuril Adapter (123347)

Ofer Reany 1
  1. Department of Natural Sciences, The Open University of Israel, Ra'anana, ISRAEL, Israel

Pores containing molecular adapters provide internal selective binding sites, thereby allowing the stochastic sensing of analytes.1 When an analyte blocks the engineered protein pore, it inhibits the ionic current through the pore with a characteristic signature, notably the extent of current block and its mean duration, providing single-molecule sensing that reveals the analyte‘s identity and concentration.

Wild-type and engineered α-Hemolysin (αHL) nanopores have been used to mediate the stochastic sensing of various analytes. For example, electrical recording with cyclodextrins as molecular adapters has permitted the stochastic sensing of adamantane-based guests, nucleotides, ribonucleoside diphosphates (rNDPs), 2′-deoxyribonucleoside 5′-monophosphates (dNMP), chiral drug molecules, and other therapeutics. Similarly, hydrophilic cyclic peptide adapters allowed the sensing of polyanion blockers, and cucurbit[6]uril mediated the sensing of tetrahydrofuran in the presence of monovalent cations.

Surprisingly, the use of single-molecule sensing to monitor inorganic anions of biological and environmental interest has never been reported, even though several strategies, including mutagenesis, metal ions chelated to DNA or peptides and polyamine-decorated cyclodextrin, have allowed the stochastic sensing of metal ions such as Ag(I), Cu(II) and Cd(II). More specifically, chloride sensing by nanopore technologies has never been applied in biomedicine, although stochastic sensing of chloride anions is highly desirable, considering their significant role in diverse cellular processes.2

Herein we demonstrate the first successful chloride detection mediated by WT-αHL equipped with an anion-binding adapter, a hydrophilic semiaza-bambus[6]uril containing six N-hydroxypropyl groups. We used single-channel recording to monitor ionic current blockades corresponding to non-covalent interactions between the bambusuril and the stem domain of the WT-αHL pore. The anion binding site within the adapter allows the detection of chloride anions, thus converting a non-selective pore into a chloride anion sensor. Furthermore, current recording allowed investigation of the kinetics between all components of the system.3

  1. 1. (a) Gu, L.-Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature, 1999, 398, 686-690. (b) Mayer, S. F.; Cao, C.; Dal Peraro, M. Biological nanopores for single-molecule sensing. iScience, 2022, 25, 104145.
  2. 2. Zajac, M.; Chakraborty, K.; Saha, S.; Mahadevan, V.; Infield, D. T.; Accardi, A.; Qiu, Z.; Krishnan, Y. What biologists want from their chloride reporters – a conversation between chemists and biologists. J. Cell Sci., 2020, 133, 1-13.
  3. 3. Reany, O, Romero-Ruiz, M. Khurana, R., Mondal, P.; Keinan, E., Bayley, H. Stochastic sensing of chloride anions using α-hemolysin pore with semiaza-Bambusuril adapter. Angew. Chem. Int. Ed., 2024, 136, e202406719.