Single-molecule sensing of peptides and nucleic acids by engineered aerolysin nanopores

Abstract

Nanopore sensing is a powerful single-molecule approach for the detection of biomolecules. Recent studies have demonstrated that aerolysin is a promising candidate to improve the accuracy of DNA sequencing and to develop novel single-molecule proteomic strategies. However, the structure–function relationship between the aerolysin nanopore and its molecular sensing properties remains insufficiently explored. Herein, a set of mutated pores were rationally designed and evaluated in silico by molecular simulations and in vitro by single-channel recording and molecular translocation experiments to study the pore structural variation, ion selectivity, ionic conductance and capabilities for sensing several biomolecules. Our results show that the ion selectivity and sensing ability of aerolysin are mostly controlled by electrostatics and the narrow diameter of the double β-barrel cap. By engineering single-site mutants, a more accurate molecular detection of nucleic acids and peptides has been achieved. These findings open avenues for developing aerolysin nanopores into powerful sensing devices.

Sensing with Nanopores and Aptamers: A Way Forward

“…This pore enables highly reproducible DNA translocation experiments. Some other biological nanopores [ 33 ] commonly used for translocation of nucleic acids, small peptides or unfolded proteins are Outer membrane protein G OmpG [ 34 ] (with an internal diameter of 1.3 nm), Mycobacterium smegmatis porin A MspA [ 35 ] (1.2 nm) and Aerolysin AeL [ 36 , 37 ] (1.0 nm). However, for sensing larger molecules such as proteins, different biological nanopores with a wider diameter are also used, such as Cytolysin A ClyA [ 38 , 39 ] (diameter 3.3 nm) and phi29 motor pores [ 40 , 41 ] (3.6 nm).…”

Multisensor Systems and Arrays for Medical Applications Employing Naturally-Occurring Compounds and Materials

“…The MSA provided the LOD of 1 cfu/mL for bacteria detection and sensitivity to DNA in the range of 0.002–200 μM. The recent development of the analytical platforms on the base of artificial or biological pores with nanoscale dimensions was proved to be a successful strategy for the detection of molecules the compatible size, as DNA, RNA, peptides, proteins and polysaccharides [ 156 ]. The protein aerolysin, produced by bacteria as a β-pore-forming toxin, was applied in the electrochemical detection of botulinum toxin type B by discriminating enzymatically cleaved peptides from a synaptic protein synaptobrevin 2 derivative [ 157 ].…”

Single‐Molecule Study of Peptides with the Same Amino Acid Composition but Different Sequences by Using an Aerolysin Nanopore

“…[78] The two main sensing regions of aerolysin nanopore that containing two critical positively charged amino acids of R220 and K238, respectively, were identified by combining the mutant nanopore experiments and molecular dynamics (MD) simulations. [64,79] The aerolysin-analyte interactions at these sensing regions can augment the sensitivity of the targeted analyte readout. Here, taking advantage of the high sensitivity of the aerolysin nanopore, [62] we sought to identify even more similar analytes.…”