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TitleBase modifications reshape RNA folding landscapes and structure-function relationships in synthetic and natural RNAs.
Journal, issue, pagesRes Sq, Year 2025
Publish dateMay 29, 2025
AuthorsEwan McRae / Deepak Yadav / Haoyun Yang / Sukyeong Lee /
PubMed AbstractModified bases such as 5-methylcytosine (5mC) and N1-methyl-pseudouridine (N1Ψ) are widely used to reduce the immunogenicity of and enhance the stability and translational efficiency of therapeutic ...Modified bases such as 5-methylcytosine (5mC) and N1-methyl-pseudouridine (N1Ψ) are widely used to reduce the immunogenicity of and enhance the stability and translational efficiency of therapeutic RNAs, yet their effects on RNA 3D structure remain poorly understood. Here, we investigate how these base modifications influence the folding and function of structured RNAs using both an engineered RNA origami nanostructure and a naturally occurring ribozyme. We show that modified bases impair kinetic maturation of RNA nanostructures and promote alternative folding topologies via stabilization of noncanonical stacking arrangements. Cryo-EM and FRET experiments reveal that this conformational shift is driven not by changes in base pairing, but by altered stacking energetics at key junctions. Our findings highlight the need to consider structural consequences when using base modifications and offer design principles for the development of functional, structured RNAs in synthetic biology and therapeutic applications.
External linksRes Sq / PubMed:40502763 / PubMed Central
MethodsEM (single particle)
Resolution4.9 - 12.1 Å
Structure data

EMDB-70035: 5mC-6HBC-monomer RNA
Method: EM (single particle) / Resolution: 7.9 Å

EMDB-70036: 5mC-6HBC-Type-1-dimer RNA
Method: EM (single particle) / Resolution: 12.1 Å

EMDB-70037: 5mC-6HBC-TC-monomer RNA
Method: EM (single particle) / Resolution: 8.0 Å

EMDB-70038: 5mC-TC-6HBC Type-2 dimer RNA
Method: EM (single particle) / Resolution: 11.3 Å

EMDB-70039: N1Y-TC-6HBC type-1 dimer RNA
Method: EM (single particle) / Resolution: 11.4 Å

EMDB-70040: N1Y-TC-6HBC type-2 dimer RNA
Method: EM (single particle) / Resolution: 12.0 Å

EMDB-70041: 5mC Tetrahymena Ribozyme
Method: EM (single particle) / Resolution: 4.9 Å

Source
  • synthetic construct (others)
  • Tetrahymena (eukaryote)

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