Madhanagopal, B. R.; Patel, A.; Talbot, H.; Geary, J. A.; Ganesh, K. N.; Chandrasekaran, A. R.

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 a 14 bp duplex DNA arm and two PNA/DNA hybrid arms. We show that the PNA/DNA mesojunction can be assembled in magnesium-free buffers containing low concentrations of calcium or sodium salts. PNA/DNA hybrid junctions and mesojunctions showed higher thermal stability compared to the DNA versions. Further, PNA/DNA hybrid junctions assembled in sodium exhibited higher nuclease resistance against DNase I. Our results pave the way for PNA/DNA hybrid three-way junctions to be integrated into complex DNA nanostructure designs to improve their structural and enzymatic stability.