Protein nanopores offer an inexpensive, label-free method of analysing single oligonucleotides. The sensitivity of the approach is largely determined by the characteristics of the pore-forming protein employed, and typically relies on nanopores that have been chemically modified or incorporate molecular motors. Effective, high-resolution discrimination of oligonucleotides using wild-type biological nanopores remains difficult to achieve. Here, we show that a wild-type aerolysin nanopore can resolve individual short oligonucleotides that are 2 to 10 bases long. The sensing capabilities are attributed to the geometry of aerolysin and the electrostatic interactions between the nanopore and the oligonucleotides. We also show that the wild-type aerolysin nanopores can distinguish individual oligonucleotides from mixtures and can monitor the stepwise cleavage of oligonucleotides by exonuclease I.

Nanopore techniques offer the possibility to study biomolecules at the single-molecule level in a low-cost, label-free and high-throughput manner. By analyzing the level, duration and frequency of ionic current blockades, information regarding the structural conformation, mass, length and concentration of single molecules can be obtained in physiological conditions. Aerolysin monomers assemble into small pores that provide a confined space for effective electrochemical control of a single molecule interacting with the pore, which significantly improves the temporal resolution of this technique. In comparison with other reported protein nanopores, aerolysin maintains its functional stability in a wide range of pH conditions, which allows for the direct discrimination of oligonucleotides between 2 and 10 nt in length and the monitoring of the stepwise cleavage of oligonucleotides by exonuclease I (Exo I) in real time. This protocol describes the process of activating proaerolysin using immobilized trypsin to obtain the aerolysin monomer, the construction of a lipid membrane and the insertion of an individual aerolysin nanopore into this membrane. A step-by-step description is provided of how to perform single-oligonucleotide analyses and how to process the acquired data. The total time required for this protocol is ∼3 d.

An aerolysin nanopore is employed as a sensitive tool for single-molecule analysis of short oligonucleotides (≤10 nucleotides), poly(ethylene glycol) (PEGs), peptides, and proteins. However, the direct analysis of long oligonucleotides with the secondary structure (e.g., G-quadruplex topology) remains a challenge, which impedes the further practical applications of the aerolysin nanopore. Here, a simple and applicable method of aerolysin nanopore is presented to achieve a direct analysis of structured oligonucleotides that are extended to 30 nucleotides long by a cation-regulation mechanism. By regulating the cation type in electrolyte solution, the structured oligonucleotides are unfolded into linear form which ensures the successive translocation. The results show that each model oligonucleotide of 5'-(TTAGGG) -3' can produce a well-resolved current blockade in its unfolded solution of MgCl . The length between 6 and 30 nucleotides long of model oligonucleotides is proportional to the duration time, showing a translocation velocity as low as 0.70-0.13 ms nt at +140 mV. This method exhibits an excellent sensitivity and a sufficient temporal resolution, provides insight into the aerolysin nanopore methodology for genetic and epigenetic biosensing, making aerolysin applicable in practical diagnosing with long and structured nucleic acids.

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Aerolysin‐based direct analysis of oligonucleotides with secondary structure (e.g., G‐quadruplex) remains a challenge. In article number https://doi.org/10.1002/smll.201704520, Yi‐Lun Ying, Yi‐Tao Long, and co‐workers present a strategy employing an aerolysin nanopore to analyze structured oligonucleotides that are extended to 30 bases in length by a cation‐regulation mechanism. This method can in principle be applied to genetic and epigenetic biosensing, making it practical for diagnostics.