A General Strategy of Aerolysin Nanopore Detection for Oligonucleotides with the Secondary Structure

Abstract 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)_n‐3′ can produce a well‐resolved current blockade in its unfolded solution of MgCl₂. 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⁻¹ 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.