Probing Structural Variants of Irregular DNA G-Tracts (<i>N</i> ≤ 2) Using MspA Nanopores

Guanine-rich DNA sequences with short G-tracts (<i>n</i> ≤ 2) are highly prevalent and abundant in the human genome, some of which are found to be associated with diseases (Maity et al. <i>Nucleic Acids Res</i>. 2020, 48 (6), 3315-3327). Unlike conventional G-quadruplexes with three or more folded layers, these sequences with G2 tracts featuring two bilayered blocks remain largely unexplored. Here, we employed nanopore experiments and all-atom molecular dynamics simulations to investigate the unwinding strengths and dynamics of these bilayered blocks. Our results demonstrated that in an electric field, the tumor-targeting element AS1411, along with its derivatives AT11 and Z-G4, strongly interacted with the M2-MspA nanopore, resulting in at least two distinct populations (types I and II events) characterized by different current blockage fractions and dwell times. Despite AS1411 being well characterized with up to eight secondary structures by nuclear magnetic resonance spectroscopy, our nanopore experiments revealed only two populations. This could be reasonably explained by (i) reversible docking with high rigidity and (ii) strand separation and translocation. Notably, a new event type (type III) for Z-G4 suggested reduced susceptibility in the last layer, contributing to its increased rigidity. Furthermore, voltage-dependent dynamics revealed that Z-G4 exhibited extended dwell times for docking and partial unwinding, unlike AT11. Our in-solution nanopore experiments and MD simulation results would benefit toward understanding the folding principles of complicated structural variants by sequences consisting of multiple short G-tracts, paving the way for the rapid identification of similar-sequence nucleic acid aptamers in molecular diagnostics and targeted therapies.