Entropy-Driven One-Step Formation of Phi29 pRNA 3WJ from Three RNA Fragments

[Image: see text] The emerging field of RNA nanotechnology necessitates creation of functional RNA nanoparticles but has been limited by particle instability. It has been shown that the three-way junction of bacteriophage phi29 motor pRNA has unusual stability and can self-assemble from three fragments with high efficiency. It is generally believed that RNA and DNA folding is energy landscape-dependent, and the folding of RNA is driven by enthalpy. Here we examine the thermodynamic characteristics of the 3WJ components as 2′-fluoro RNA, DNA, and RNA. It was seen that the three fragments existed either in 3WJ complex or as monomers, with the intermediate of dimers almost undetectable. It seems that the three fragments can lead to the formation of the 3WJ complex efficiently within a rapid time. A low dissociation constant (apparent K(D)) of 11.4 nM was determined for RNA, inclusion of 2′-F pyrimidines strengthened the K(D) to 4.5 nM, and substitution of DNA weakened it to 47.7 nM. The ΔG°(37), were −36, −28, and −15 kcal/mol for 3WJ(2′-F), 3WJ(RNA), and 3WJ(DNA), respectively. It is found that the formation of the three-component complex was governed by entropy, instead of enthalpy, as usually found in RNA complexes.