Mechanical Constraints Promote Noncanonical Base-Pairing Interactions

Mechanical perturbations profoundly influence nucleobase interactions in cells, serving as key modulators of fundamental cellular processes such as transcriptional regulation. However, their precise roles remain elusive due to methodological limitations. Here, we have developed a DNA origami-based nanobender system that probes base-base interactions under mechanical constraints. This system imposes tunable geometric and mechanical constraints on single-stranded DNA/RNA by pulling its two ends together while adjusting its contour length, and enables the assessment of weak interactions between bases through the detection of DNA origami dimerization. Combining experiments and molecular dynamics simulations, we speculate that mechanical constraints promote stacking-driven noncanonical base-pairing via entropy compensation and favorable nucleobase orientation (hydrophobic exposure), advancing our understanding of base recognition diversity. Customizable nanobender parameters allow investigation of the effects of geometric and mechanical constraints on hybridization thermodynamics across DNA-DNA, RNA-RNA, DNA-RNA, and epigenetically modified nucleotides. Notably, we suggest that entropy modulation mediated by mechanical constraints enables fine-tuning of binding free energy, thereby expanding material assembly applications while conferring environmental sensitivity.