Eliminating the Interference of Neighboring Nucleobases in Aerolysin for Nanopore Sequencing

Biological nanopores have revolutionized DNA sequencing with their incredible advantages of long reads, high throughput, portability, and low material requirement. Despite numerous improvements, base calling remains challenging due to the influence of neighboring nucleobases at the reading site. One direction of development is exploring or designing new nanopores to improve base calling accuracy. Aerolysin is emerging as a powerful candidate, which can identify the subtle differences of detected molecules. However, not many studies focus on translating these advantages into DNA sequencing. Here, we used streptavidin to immobilize ssDNA inside an engineered aerolysin channel, which allows us to investigate its sensing regions, and the neighboring bases influence. By increasing the voltage, we eliminate the neighboring influence and reduce the k-mer size to one nucleobase. These findings hold great potential for improving the accuracy of nanopore DNA sequencing as well as aerolysin nanopore-based sequencing for other polymers.