Conserved RNA architecture organizes Mg²⁺-mediated RNA assemblies into functionally competent structural states

Higher-order RNA assemblies have emerged as an intrinsic property of RNA under Mg²⁺ conditions, raising the question of whether conserved RNA architecture contributes to their organization and functional competence. Here, we investigated the contribution of conserved RNA architecture to higher-order RNA assembly using conserved PTC-derived RNA fragments representing the A- and P-site regions of the large ribosomal subunit. These RNA fragments exhibited distinct Mg²⁺-dependent condensation behavior and cooperatively formed dynamic higher-order RNA assemblies. Replacing one conserved PTC-derived fragment with an architectural-control RNA altered RNA assembly behavior and failed to support functional competence in modified ribosomal fragment assays. Primitive peptidyl transfer chemistry serves as a functional readout of higher-order RNA organization. Accordingly, our findings suggest that conserved RNA architecture organizes higher-order RNA assemblies into functionally competent structural states.