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Base modifications reshape RNA folding landscapes and structure-function relationships in synthetic and natural RNAs
Ewan McRae1, Deepak Yadav1, Haoyun Yang1
1Houston Methodist Research Institute.
Research Square
|June 12, 2025
Summary
Modified bases like 5-methylcytosine and N1-methyl-pseudouridine impact RNA 3D structure, affecting folding and function. These modifications can stabilize noncanonical arrangements, influencing the design of therapeutic and synthetic RNAs.
Area of Science:
- Biochemistry
- Structural Biology
- Synthetic Biology
Background:
- Modified bases such as 5-methylcytosine (5mC) and N1-methyl-pseudouridine (N1Ψ) are crucial for therapeutic RNA applications, enhancing stability and reducing immunogenicity.
- However, the impact of these modifications on the three-dimensional structure and folding of RNA remains largely uncharacterized.
Purpose of the Study:
- To investigate the influence of 5mC and N1Ψ base modifications on the folding pathways and structural conformations of engineered and natural structured RNAs.
- To elucidate the mechanistic basis for structural alterations induced by modified bases in RNA.
Main Methods:
- Utilized an engineered RNA origami nanostructure and a naturally occurring ribozyme as model systems.
- Employed Cryo-electron microscopy (Cryo-EM) and Förster resonance energy transfer (FRET) experiments to analyze RNA structures and dynamics.
- Assessed kinetic maturation pathways and folding topologies of modified and unmodified RNA constructs.
Main Results:
- Modified bases were found to impede the kinetic maturation of RNA nanostructures.
- These modifications promote alternative folding pathways by stabilizing noncanonical base stacking arrangements.
- Structural analysis revealed that altered stacking energetics, not base pairing changes, drive the observed conformational shifts.
Conclusions:
- Base modifications significantly influence RNA folding kinetics and final structure, stabilizing noncanonical stacking interactions.
- Consideration of these structural consequences is essential for the rational design of modified therapeutic and synthetic RNAs.
- Findings provide design principles for developing functional, structured RNAs for synthetic biology and therapeutic purposes.
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