Base modifications shift tertiary structure and activity in synthetic RNA origami and a natural ribozyme

Modified nucleotide bases like 5-methylcytosine (m5C) and N1-methyl-pseudouridine (m1Ψ) are widely used to enhance stability and reduce immunogenicity in therapeutic RNAs, yet their impact on RNA tertiary structure remains unclear. Here we investigate how these modifications influence folding and function in both a synthetic RNA origami nanostructure and the natural Tetrahymena ribozyme. Using cryo-EM, FRET, and biochemical assays, we find that modified bases impede proper maturation of RNA origami by stabilizing alternative coaxial stacking at key junctions, leading to dimerization. In the ribozyme, modifications shift the equilibrium between open and closed conformations, altering catalytic activity in a temperature-dependent manner. These effects arise primarily from changes in base-stacking energetics rather than base pairing. Our findings reveal that base modifications reshape RNA folding landscapes and structure–function relationships, underscoring the need to consider structural consequences when designing modified RNAs for synthetic biology and therapeutic applications.