Studying RNA-only molecules such as catalytic ribozymes and the frameshifting stimulation element (FSEs) of coronaviruses can aid in the development of potential drug targets and therapies, but capturing the structures of these “naked” ribonucleic acids is challenging without proteins and other molecules to hold them in place. Now, researchers from Stanford University and the Department of Energy’s SLAC National Accelerator Laboratory at Stanford have developed a system combining computational tools and single-particle cryogenic electron microscopy (cryo-EM) to uncover the structure of a well-known ribozyme in unprecedented resolution.
The researchers built upon an existing Stanford-developed RNA pipeline called Ribosolve, which combines cryo-EM imaging advances, computational tools and chemical mapping tools to quickly solve the structures of RNAs in unprecedented detail. The Ribosolve method had previously been used to determine the 3D structures of the Tetrahymena thermophila ribozyme and 10 other RNA molecules in better than 10 Å resolution. The Tetrahymena ribozyme was the first catalytic RNA molecule ever discovered about 40 years ago, and has since been studied extensively by scientists hoping to unlock many of the secrets that still remain about RNA.
An animation depicts the Tetrahymena
ribozyme as a long, narrow ribbon with many twists and folds, and reveals how one of its ends moves while binding to two smaller RNA molecules. The tail end of the ribozyme (orange) swings into a new position along with a few more nucleotides (purple). Then the tail and its extension bind with the two RNA molecules (green and blue) , forming a double helix. This mimics one of the key steps in the process where the ribozyme cuts itself out of the middle of a strand of RNA and splices the two loose ends back together. The animation is based on images made at SLAC and Stanford that reveal the molecular structures in unprecedented detail. Credit: Grigore Pintilie, Stanford University
In their most recent study, the team improved upon their methods and were successful in solving the Tetrahymena ribozyme structures at a resolution of 3.1 Å, forming a more detailed picture of the RNA-only molecule than ever before and demonstrating Ribosolve’s value as a tool to better understand RNA structures of interest. The enhanced method allowed the researchers to uncover previously unresolved structures and unforeseen interactions within the ribozyme. The research, led by Wah Chiu of SLAC/Stanford and Rhiju Das of Stanford, was published in Nature.
“I do think the Ribosolve pipeline has the potential to transform our understanding of these molecules, and maybe our ability to develop medicines, too,” said Das. “This couldn’t have happened anywhere else. Having access to world-class cryo-EM instruments was key, along with meeting someone like Wah who shared our intuition that this could be important.”
The researchers also used Ribosolve to study the FSE of SARS-CoV-2 and the results of that research have been posted as a preprint pending peer review. The team believes the detailed insights into the 3D structures of viral RNA molecules gained through their RNA pipeline could help in the engineering of drugs that can effectively target and inhibit these molecules.
“We don’t know what the next pandemic virus will be, but we’re pretty confident it will be a single-strand RNA virus transmitted from animals to humans, and it will likely have a few bits of RNA that resist mutation,” said Das. “With this accelerated system we’ve developed, it now seems feasible to study viruses found in humans or animals, look for those conserved bits, quickly determine their 3D RNA structures and develop antivirals against them.”