New Imaging Method Unlocks Secrets of RNA Folding Dynamics

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 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 the 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 link to video below).

"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.