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

Abstract

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.

Nanopore-Based Single-Molecule Investigation of Cation Effect on the i-Motif Structure

“…Furthermore, the histograms of logarithm dwell times for all events in 1 M LiCl, NaCl, KCl, and CsCl showed two peaks, characterized by i-motif (vermilion) and ss-DNA (blue), whereas the histograms of logarithm dwell times for all events in 1 M MgCl 2 and BaCl 2 showed only the peak of ss-DNA (Figure S4). In the solutions of 1 M LiCl, NaCl, KCl, and CsCl, the logarithmic dwell times for long-lived events were 3.27 ± 0.04, 3.30 ± 0.06, 2.90 ± 0.02, and 2.67 ± 0.03, respectively, and the relative current blockages for long-lived events were 0.90 ± 0.01, 0.87 ± 0.01, 0.86 ± 0.01, and 0.88 ± 0.01, respectively, which were consistent with the i-motif characteristic event 41,42 (Figure S4). However, the magnitude of electrophoretic forces on the imotif varied across different electrolyte solutions, as evidenced by the differing frequencies of all events.…”