3D Snapshots Unveil the Intricate Dance of RNA Folding
In a groundbreaking development, researchers from the United States and Denmark have successfully captured 3D images of individual RNA nanoparticles in the midst of their folding process. Utilizing a cutting-edge electron microscopy technique, the team has unlocked new insight into the intricate folding dance leading to the final shape of RNA molecules.
The flexibility of RNA makes it notoriously challenging to study, as its structure can shift into numerous forms depending on environmental conditions. Traditional imaging methods, such as cryo-electron microscopy (cryo-EM) single-particle averaging (SPA) analysis, rely on averaging data from thousands of selected molecules with common shapes, making it difficult to capture the unique shapes of individual RNA molecules.
In a study published in Nature Communications, researchers from the Molecular Foundry at Lawrence Berkeley National Laboratory and the Interdisciplinary Nanoscience Center and the Department of Molecular Biology and Genetics at Aarhus University explored the folding process of flexible RNA molecules. They employed an innovative technique capable of studying the 3D shape of individual molecules without averaging. This technique builds on advanced Individual-Particle cryo-Electron Tomography (IPET), a specialized approach that focuses on single molecule 3D imaging in cryo-preserved samples (see figure and fact box below). Previously, this technique has been used to study how nucleosomes fold DNA and induce phase transitions.
Historically, scientists believed that obtaining 3D images from a single molecule was impossible due to weak signals. "It was considered a dead-end method since 1970s," said Gang Ren, a staff scientist at the Molecular Foundry, who co-led the research alongside Ebbe Andersen from Aarhus University.
In the current study, the researchers used IPET to study RNA origami – artificially structured RNA molecules engineered to fold into specific nanoscale shapes. Ebbe Andersen and colleagues had previously used the cryo-EM SPA method to study the 3D structure of RNA origami but the folding process remained elusive. IPET allowed the researchers to capture a snapshot of RNA’s folding landscape through capturing molecules in various stages of folding, from immature states to their optimal shape. The researchers were able to observe a folding trap and a shift to a more compact form, enabling creation of a “movie” depicting RNA’s dynamic folding process (see video).
The video shows the process of focusing on a single molecule to reveal its 3D shape using the IPET method. It then shows the analysis of 120 particles and an animation to illustrate the dynamic and intricate folding process of RNA origami particles.
“The IPET technique provides us with a more dynamic view of the molecular world. It is our hope that this insight will enable us to engineer the folding of more effective RNA vaccines and dynamic sensors for molecular medicine”, explains Ebbe Andersen.
Study type:
Experimental Molecular Biology
External funding:
This project has received funding from the following research grants: the US National Institutes of Health grants of R01HL115153, R01GM104427, R01MH077303, and R01DK042667 (G.R., J.L., and M.Z.); Independent Research Fund Denmark grant 9040-00425B (E.S.A. and E.K.S.M.); Canadian Natural Sciences and Engineering Research Council grant 532417 (EKSM); European Research Council (ERC) Consolidator grant 683305 (E.S.A., C.G., E.K.S.M.), and Novo Nordisk Foundation Ascending Investigator grant 0060694 (E.S.A. and C.G.).
Conflicts of interest:
The authors declare no competing interests.
Link to the scientific article:
"Non-averaged single-molecule tertiary structures reveal RNA self-folding through individual-particle cryo-electron tomography" by Jianfang Liu, Ewan K. S. McRae, Meng Zhang, Cody Geary, Ebbe Sloth Andersen, and Gang (Gary) Ren
https://www.nature.com/articles/s41467-024-52914-1
Contact information:
Staff Scientist Gang (Gary) Ren
The Molecular Foundry
Lawrence Berkeley National Laboratory
Email: gren@lbl.gov
Associate Professor Ebbe Sloth Andersen
Interdisciplinary Nanoscience Center
Department of Molecular Biology and Genetics
Aarhus University
Email: esa@inano.au.dk