BPS2026 – Peptide nucleic acid (PNA) at the crossroads of DNA

Three-way junctions are simple and fundamental structural motifs that impart the typical branching property in most DNA nanostructures. While conventional three-way DNA junctions are well-known, mesojunctions are relatively unexplored. Here, we report the synthesis of peptide nucleic acid (PNA)/DNA hybrid three-way conventional and mesojunctions, containing one 14 bp-long duplex DNA arm and two 14 bp-long PNA/DNA hybrid arms. Incorporating PNA into the junction enables assembly in magnesium-free buffers with low concentrations of calcium or sodium and significantly enhances thermal and nuclease stability compared to all-DNA junctions. Molecular dynamics simulations reveal how the neutral PNA backbone influences base stacking across the junction. These findings establish hybrid DNA/PNA junctions as robust structural motifs that expand the design space of nucleic-acid nanotechnology and enable the development of stable, functional architectures for therapeutic and biomolecular engineering applications.