(Nanowerk News) 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.
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.
Left side of figure shows the process of individual-particle cryo-electron tomography (IPET) where RNA samples are frozen on a grid, then imaged by tilting the sample at different angles. The images are then used to reconstruct the 3D shape of the particle. Right side of figure shows the 3D shape of individual RNA molecules. (Image: Aarhus University)
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.
