Paranemic Cohesion of DNA under Isothermal Conditions

Recent progress in DNA nanotechnology has shown the isothermal assembly of several DNA nanostructures. Isothermal assembly allows DNA nanostructure construction in a variety of ions while simplifying DNA nanotechnology by avoiding the need for thermal cyclers and expands utility by enabling attachment of guest biomolecules on DNA nanostructures at ambient or physiological temperatures. The paranemic crossover (PX) DNA motif has been used in the construction of DNA nanostructures, paranemic cohesion has been used to connect DNA structures as an alternate to sticky end cohesion, and PX DNA has also been implied to have a biological role in homology recognition. In that context, here we demonstrate the successful isothermal assembly of the PX DNA motif in magnesium (Mg²⁺), calcium (Ca²⁺), and strontium (Sr²⁺) at 20 and 37 °C. Using isothermal titration calorimetry, we show that interhelix hybridization of half-PX molecules is favored at higher temperatures, with a heat capacity (ΔC_p) of -1.9 kcal/mol·K. To demonstrate a key advantage of isothermal assembly, we show that PX molecules can be designed to contain thrombin-specific aptamers for binding one or two thrombin molecules site specifically in an entirely isothermal procedure. Our work extends isothermal assembly and the use of different counterions for complex DNA motifs while demonstrating the attachment of guest molecules at constant temperatures.