Nanopore-Based Single-Molecule Investigation of DNA Sequences with Potential to Form i-Motif Structures

i-Motifs are DNA secondary structures present in cytosine-rich sequences. These structures are formed in regulatory regions of the human genome and play key regulatory roles. The investigation of sequences capable of forming i-motif structures at the single-molecule level is highly important. In this study, we used α-hemolysin nanopores to systematically study a series of DNA sequences at the nanometer scale by providing structure-dependent signature current signals to gain in-sights into the i-motif DNA sequence and structural stability. Increasing the length of the cytosine tract in a range of 3-10 nucleobases resulted in a longer translocation time through the pore, indicating improved stability. Changing the loop sequence and length in the sequences did not affect the formation of the i-motif structure but changed its stability. Importantly, the application of all-atom molecular dynamics simulations revealed the structural morphology of all sequences. Based on these results, we postulated a folding rule for i-motif formation, suggesting that thousands of cytosine-rich sequences in the human genome might fold into i-motif structures. Many of these were found in locations where structure formation is likely to play regulatory roles. These findings provide insights into the application of nanopores as a powerful tool for discovering potential i-motif-forming sequences and lay a foundation for future studies exploring the biological roles of i-motifs.