Mechanism of three-component collision to produce ultrastable pRNA three-way junction of Phi29 DNA-packaging motor by kinetic assessment

RNA nanotechnology is rapidly emerging. Due to advantageous pharmacokinetics and favorable in vivo biodistribution, RNA nanoparticles have shown promise in targeted delivery of therapeutics. RNA nanotechnology applies bottom-up assembly, thus elucidation of the mechanism of interaction between multiple components is of fundamental importance. The tendency of diminishing concern about RNA instability has accelerated by the finding of the novel thermostable three-way junction (3WJ) motif of the phi29 DNA-packaging motor. The kinetics of these three components, each averaging 18 nucleotides (nt), was investigated to elucidate the mechanism for producing the stable 3WJ. The three fragments coassembled into the 3WJ with extraordinary speed and affinity via a two-step reaction mechanism, 3WJ_b + 3WJ_c ↔ 3WJ_bc + 3WJ_a ↔ 3WJ_abc. The first step of reaction between 3WJ_b and 3WJ_c is highly dynamic since these two fragments only contain 8 nt for complementation. In the second step, the 3WJ_a, which contains 17 nt complementary to the 3WJ_bc complex, locks the unstable 3WJ_bc complex into a highly stable 3WJ. The resulting pRNA-3WJ is more stable than any of the dimer species as shown in the much more rapid association rates and slowest dissociation rate constant. The second step occurs at a very high association rate that is difficult to quantify, resulting in a rapid formation of a stable 3WJ. Elucidation of the mechanism of three-component collision in producing the ultrastable 3WJ proves a promising platform for bottom-up assembly of RNA nanoparticles as a new class of anion polymers for material science, electronic elements, or therapeutic reagents.