Nanopore Discriminates Watson–Crick and Hoogsteen Hydrogen Bonds in Multiple DNA Contexts

Watson–Crick and Hoogsteen hydrogen bonds aid the formation of highly ordered structures in genomic DNA that dynamically govern genetic modes such as gene regulation and replication. Hence, measuring and distinguishing these two types of hydrogen bonds in different DNA contexts are essential for understanding DNA architectures. However, due to their transient nature and minimal structure differences at the sub-nanometer scale, differentiating Watson–Crick hydrogen bonds from Hoogsteen hydrogen bonds is difficult. Relying on nanopore technology, we successfully discriminated the two types of hydrogen bonds in multiple DNA contexts in the presence of epigenetic modification, changes in DNA structures, and proton strength in the environment. Our results indicate that Watson–Crick and Hoogsteen hydrogen bonds show different susceptibilities to changes in physicochemical characteristics that matter in stabilizing DNA hydrogen bonds. This work provides insight into the features of hydrogen bonds at the nanoscale and may benefit profiling complex DNA architectures by measuring subtle structural changes.