RNA in action: filming a ribozyme’s self-assembly

Researchers have visualised, in unprecedented detail, how a large RNA molecule assembles itself into a functional machine. This work provides new insights into RNA folding and misfolding, and helps set the stage for RNA design and engineering. 

RNA is a central biological molecule, now widely harnessed in medicine and nanotechnology. Like proteins, RNA’s function is often derived from its three-dimensional structure. In a new study, researchers from the Marcia lab (EMBL Grenoble and Uppsala University), the Topf lab (LIV/UKE/CSSB) and the De Vivo lab (Istituto Italiano di Tecnologia) have recorded how a large RNA molecule folds, flexes, and assembles itself into a functional biological machine, almost frame by frame. 

Using a range of state-of-the-art techniques — cryo-electron microscopy (cryo-EM), small-angle X-ray scattering (SAXS), RNA biochemistry and enzymology, image processing, and molecular simulations — the scientists captured the dynamic process by which a self-splicing ribozyme folds into its functional structure. 

Traditional cryo-EM workflows often struggle when the sample is structurally heterogeneous, flexible or dynamic, such as RNA molecules. To avoid these challenges, Mauro Maiorca, a postdoctoral scientist in the Topf lab, developed a novel image-processing method to help analyse Cryo-EM data from several hundred thousand noisy images. This method groups particles into consistent conformational classes, enhancing the local resolution of functionally important regions within each class. This enabled the team to reconstruct intermediate states that are usually invisible in static crystal structures. The atomic models built in cryo-EM density maps were refined by another postdoctoral scientist in the Topf lab, Thomas Mulvaney, who developed TEMPy-ReFF, a software tool for atomic structure refinement in cryo-EM density maps. 

The work is an excellent example of how innovative tools for robust particle classification and the density-guided refinement, which were developed at the LIV for integrative cryo-EM, are particularly powerful for resolving dynamic assemblies solved by single-particle cryo-EM. These approaches can help to gain mechanistic insights into viral processes, such as assembly, maturation, entry and immune evasion.

The research team’s results, recently published in the scientific journal Nature Communications, deliver the most complete “molecular film” to date of an RNA building itself. The “film” also reveals how RNA avoids misfolded, non-functional states known as kinetic traps.

This work sets the stage for RNA design and engineering — guiding how future biotechnologies might script RNA molecules to fold correctly for use in therapeutics or nanobiotechnology. For RNA viruses in particular, function depends on specific conformations, including long-range RNA–RNA interactions in replication and packaging. Understanding folding pathways and non-productive kinetic traps may reveal new intervention points and enable more stable, predictable RNA-based vaccines.

Original Publication

Jadhav, S., Maiorca, M., Manigrasso, J., Saha, S., Rakitch, A., Muscat, S., Mulvaney, T., De Vivo, M., Topf, M., Marcia, M. (2025) Dynamic assembly of a large multidomain ribozyme visualized by cryo-electron microscopy. Nat Commun 16, 10195. doi.org/10.1038/s41467-025-65502-8