Mg²⁺ Ions Regulating 3WJ-PRNA to Construct Controllable RNA Nanoparticle Drug Delivery Platforms

RNA nanotechnology has shown great progress over the past decade. Diverse controllable and multifunctional RNA nanoparticles have been developed for various applications in many areas. For example, RNA nanoparticles can participate in the construction of drug delivery nanoplatforms. Recently, a three-way junction packaging RNA (3WJ-pRNA) has been exploited for its characteristics of self-assembly and ultrahigh stability in many aspects. 3WJ-pRNA is the 3WJ part of bacteriophage φ29 pRNA and joins different components of φ29 as a linker element. In this work, we used all-atom MD simulation to study the thermal stability of 3WJ-pRNA and the underlying mechanisms. While 3WJ-pRNA can remain in its original structure without Mg²⁺ ions at room temperature, only Mg-bound 3WJ-pRNA still maintains its initial three-way junction structure at a higher temperature (T = 400 K). The Mg-free 3WJ-pRNA undergoes dramatic deformation under high temperature condition. The contribution of Mg ions can be largely attributed to the protective effect of two Mg clamps on the hydrogen bond and base stacking interactions in helices. Taken together, our results reveal the extraordinary thermal stability of 3WJ-pRNA, which can be regulated by Mg²⁺ ions. Comprehensive depictions of thermal stability of pRNA and the regulation mechanism are helpful for the further development of controllable RNA nanoparticle drug delivery platforms.