To understand what an individual RNA molecule does, its 3D structure needs to be deciphered at the level of its constituent atoms and molecular bonds. Technology used to study the structures of DNA and protein cannot be applied to RNA molecules with nearly the same effectiveness because their molecular composition and structural flexibility prevent them from easily forming crystals.

Now, a fundamentally new approach to the structural investigation of RNA molecules has been reported. The approach, which integrates RNA nanotechnology and cryo-EM approaches, is named “RNA oligomerization-enabled cryo-EM via installing kissing loops” or ROCK.

ROCK uses an RNA nanotechnological technique that allows it to assemble multiple identical RNA molecules into a highly organized structure, which significantly reduces the flexibility of individual RNA molecules and multiplies their molecular weight.

Applied to well-known model RNAs with different sizes and functions as benchmarks, the researchers used cryo-EM to show that their method enables the structural analysis of the contained RNA subunits.

This work is published in Nature Methods, in the paper, “Sub-3-Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly.

“ROCK is breaking the current limits of RNA structural investigations and enables 3D structures of RNA molecules to be unlocked that are difficult or impossible to access with existing methods, and at near-atomic resolution,” said Peng Yin, PhD, core faculty member at the Wyss Institute for Biologically Inspired Engineering at Harvard University. “We expect this advance to invigorate many areas of fundamental research and drug development, including the burgeoning field of RNA therapeutics.”

Yin’s team hypothesized that strategies that have allowed DNA to self-assemble into large structures, like DNA origami, could also be used to assemble naturally occurring RNA molecules into highly ordered circular complexes by specifically linking them together. Many RNAs fold in complex yet predictable ways, with small segments base-pairing with each other. The result often is a stabilized “core” and “stem-loops” bulging out into the periphery.

“In our approach we install ‘kissing loops’ that link different peripheral stem-loops belonging to two copies of an identical RNA in a way that allows an overall stabilized ring to be formed, containing multiple copies of the RNA of interest,” said Di Liu, PhD, a postdoctoral fellow in Yin’s lab. “We speculated that these higher-order rings could be analyzed with high resolution by cryo-EM, which had been applied to RNA molecules with first success.”

“Cryo-EM has great advantages over traditional methods in seeing high-resolution details of biological molecules including proteins, DNAs, and RNAs, but the small size and moving tendency of most RNAs prevent successful determination of RNA structures. Our novel method of assembling RNA multimers solves these two problems at the same time, by increasing the size of RNA and reducing its movement,” said Maofu Liao, PhD, associate professor of cell biology at Harvard Medical School. “Our approach has opened the door to rapid structure determination of many RNAs by cryo-EM.”

To provide proof-of-principle for ROCK, the team focused on a large intron RNA from the single-celled organism Tetrahymena, and a small intron RNA from Azoarcus, a nitrogen-fixing bacterium, as well as the FMN riboswitch. The FMN riboswitch is found in bacterial RNAs involved in the biosynthesis of flavin metabolites derived from vitamin B2. Upon binding one of them, flavin mononucleotide (FMN), it switches its 3D conformation and suppresses the synthesis of its mother RNA.

“The assembly of the Tetrahymena group I intron into a ring-like structure made the samples more homogenous, and enabled the use of computational tools leveraging the symmetry of the assembled structure. While our dataset is relatively modest in size, ROCK’s innate advantages allowed us to resolve the structure at an unprecedented resolution,” said François Thélot, PhD, Liao’s former graduate student who is currently an associate at McKinsey & Company. “The RNA’s core is resolved at 2.85 Å, revealing detailed features of the nucleotide bases and sugar backbone. I don’t think we could have gotten there without ROCK—or at least not without considerably more resources.”

Applying ROCK to the Azoarcus intron RNA and the FMN riboswitch, the team managed to identify the different conformations that the Azoarcus intron transitions through during its self-splicing process, and to reveal the relative conformational rigidity of the ligand-binding site of the FMN riboswitch.

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