Ki-Baek Jeong^a,1,2, Sang-Mook You^a,2, Jin-Sung Park^a, Ke Luo^a, In-Seong Hwang^a, Hwankyu Lee^b, Young-Rok Kim^a,*

^aGraduate School of Biotechnology & Department of Food Science and Biotechnology, Kyung Hee University, Yongin, 17104, South Korea
^bDepartment of Chemical Engineering, Dankook University, Yongin, 16891, South Korea

¹Present address: Disease Target Structure Research Center, Division of Biomedical Research, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
²K.B.J. and S.M.Y. contributed equally to this work.

^*Corresponding author.

Nanopores have been emerged as a powerful tool for analyzing the structural information and interactional properties of a range of biomolecules. The spatial resolution of nanopore is determined by the diameter and effective thickness of its constriction region, but the presence of vestibule or stem structure in protein-based nanopore could negatively affect the sensitivity of the nanopore when applied for genome sequencing and topological analysis of DNA. Recently, alpha-hederin (Ah) has been reported to form a sub-nanometer scale pore structure in lipid membrane. With the simple structure and extremely small effective thickness, the Ah nanopore was shown to discriminate four different types of nucleotides. However, identification of a certain nucleotide in a strand of DNA, which is essential for genome sequencing, remains challenging. Here, we investigated the resolving capability of Ah nanopore to discriminate few nucleotides in a strand of single-stranded DNA, and the factors determining the sensitivity of Ah nanopore. The Ah nanopore was shown to be able to identify as few as three adenosine nucleotides in a strand of poly cytidine, in which the dwell time of the additional current blockade that represents the adenosine residue was in good agreement with their physical length. We also found that the lateral tension and chain pressure generated around the nanopore were influenced by pore's diameter and played as a dependent variables to determine the geometry of nanopore's constriction as well as the spatial resolution of the Ah nanopore.