2.D.241. The Synthetic alpha-Helical Nanopore Reactor (SHNR) Family
The use of nanopores for the single-molecule sensing of folded proteins and biomacromolecules has become useful. Das et al. 2023 introduced a simplified synthetic alpha-helical transmembrane pore, pPorA, as a nanoreactor and sensor that exhibits functional versatility comparable to that of
engineered protein and DNA nanopores. The pore, built from the assembly of synthetic 40-amino-acid- long peptides, is designed to contain cysteine residues within the lumen and at the pore terminus for site-specific chemical modification probed using single-channel electrical recordings. The reaction of the pore with differently charged activated thiol reagents was studied, wherein positively charged reagents electrophoretically driven into the pore resulted in pore blocking in discrete steps upon covalent bond formation. The asymmetric blockage patterns resulting from cis and trans-side addition of reagents reveal the pore orientation in the lipid membrane. Furthermore, activated PEG thiols covalently blocked the pores over a longer duration in a charge-independent manner, establishing the large diameter and orientation of the formed pores. While the covalent binding of thiol reagents caused a drop in the pore conductance, cationic cyclic octasaccharides produced time-resolved translocation events, confirming the structural flexibility and tunability of the pores. The ability of the pore to accommodate large analytes and the considerable current amplitude variation following bond formation events are promising for developing platforms to resolve multistep chemical reactions at the single-molecule level for applications in synthetic nanobiotechnology.