We live in a world created and directed by RNA, the equally important brother of the genetic molecule DNA. In fact, evolutionary biologists hypothesize that RNA existed and self-replicated even before the appearance of DNA and the proteins encoded by it. Fast forward to today’s humans: science has revealed that less than 3% of the human genome is transcribed into messenger RNA (mRNA) molecules which, in turn, are translated into proteins. On the other hand, 82% of it is transcribed into RNA molecules with other functions, many of which still remain enigmatic.
To understand what an individual RNA molecule does, its 3D structure must be deciphered at the level of its constituent atoms and molecular bonds. Researchers have regularly studied DNA and protein molecules by turning them into regularly packed crystals that can be examined with an X-ray beam (X-ray crystallography) or radio waves (nuclear magnetic resonance). However, these techniques cannot be applied to RNA molecules with nearly the same efficiency because their molecular composition and structural flexibility prevent them from easily forming crystals.
Now a research collaboration led by Wyss Core Faculty member Peng Yin, Ph.D. at Harvard University’s Wyss Institute for Biologically Inspired Engineering, and Maofu Liao, Ph.D. at Harvard Medical School (HMS), reported on a fundamentally new approach to the structural study of RNA molecules . ROCK, as it is called, uses an RNA nanotechnology technique that allows it to assemble several identical RNA molecules into a highly organized structure, which greatly reduces the flexibility of individual RNA molecules and multiplies their molecular weight. . Applied to well-known model RNAs with different sizes and functions as references, the team showed that their method allows structural analysis of the contained RNA subunits with a technique known as cryo-electron microscopy (cryo -EM). Their lead is reported in Natural methods.
“ROCK breaks through the current limitations of RNA structural investigations and unlocks 3D structures of RNA molecules that are difficult or impossible to access with existing methods, and at near atomic resolution,” Yin said, who, with Liao, led the study. . “We expect this breakthrough to invigorate many areas of basic research and drug development, including the burgeoning field of RNA-based therapies.” Yin is also a leader of the Wyss Institute’s Molecular Robotics Initiative and a professor in the Department of Systems Biology at HMS.
Yin’s team at the Wyss Institute has developed various approaches for DNA and RNA molecules to self-assemble into large structures based on different principles and requirements, including DNA building blocks and l DNA origami. They hypothesized that such strategies could also be used to assemble natural RNA molecules into highly ordered circular complexes in which their freedom of bending and movement is severely limited by specifically binding them. Many RNAs fold in complex but predictable ways, with small segments paired with each other. The result is often a stabilized “core” and bulging “stem loops” towards the periphery.
“In our approach, we install ‘kissing loops’ that connect different peripheral stem-loops belonging to two copies of an identical RNA in such a way as to allow the formation of a global stabilized ring, containing several copies of the d ‘interest,’ said Di Liu, Ph.D., one of Yin’s group’s first two authors and postdoctoral fellow. “We hypothesized that these higher-order rings could be analyzed with high resolution by cryo-EM, which had been applied to RNA molecules with initial success.”
Imaging of stabilized RNA
In cryo-EM, many individual particles are frozen instantaneously at cryogenic temperatures to prevent further movement, then visualized with an electron microscope and using computational algorithms that compare different aspects of 2D surface projections of a particle and reconstruct its 3D architecture. . Peng and Liu teamed up with Liao and his former graduate student François Thélot, Ph.D., the study’s other co-first author. Liao and his group have made important contributions to the rapidly evolving field of cryo-EM and to the experimental and computational analysis of single particles formed by specific proteins.
“Cryo-EM has great advantages over traditional methods for viewing high-resolution detail of biological molecules, including proteins, DNAs, and RNAs, but the small size and mobile tendency of most RNAs preclude the successful determination of RNA structures. Our new method for assembling RNA multimers solves both of these problems at the same time, increasing the size of RNA and reducing its movement,” said Liao, who is also a professor Associate of Cell Biology at HMS.”Our approach has opened the door to the rapid determination of the structure of many RNAs by cryo-EM.”The integration of RNA nanotechnology and cryo-EM approaches has led the team to name their method “Cryo-EM activated by RNA oligomerization via installation of kissing loops” (ROCK).
To provide proof of principle for ROCK, the team focused on a large intron RNA from Tetrahymenea single-celled organism and a small intron RNA of Azoarcus, a nitrogen-fixing bacterium, as well as the so-called riboswitch FMN. Intron RNAs are non-coding RNA sequences scattered throughout freshly transcribed RNA sequences and must be “spliced” for mature RNA to be generated. The FMN riboswitch is found in bacterial RNAs involved in the biosynthesis of flavin metabolites derived from vitamin B2. By binding to one of them, flavin mononucleotide (FMN), it changes its 3D conformation and suppresses the synthesis of its parent RNA.
“The assembly of the Tetrahymene the group I intron in a ring-like structure made the samples more homogeneous and allowed the use of computational tools taking advantage of the symmetry of the assembled structure. While our dataset is relatively small in size, the innate advantages of ROCK allowed us to solve the structure at unprecedented resolution,” said Thélot. “The RNA core is resolved at 2.85 Å [one Ångström is one ten-billions (US) of a meter and the preferred metric used by structural biologists], revealing detailed features of the nucleotide bases and sugar backbone. I don’t think we could have done it without ROCK – or at least not without a lot more resources.”
Cryo-EM is also able to capture molecules in different states if, for example, they change their 3D conformation as part of their function. Apply ROCK to Azoarcus intron RNA and the FMN riboswitch, the team managed to identify the different conformations that the Azoarcus the intron passes through during its self-splicing process and reveals the relative conformational stiffness of the FMN riboswitch ligand-binding site.
“This study by Peng Yin and co-workers elegantly demonstrates how RNA nanotechnology can work as an accelerator to advance other disciplines. Being able to visualize and understand the structures of many naturally occurring RNA molecules could have a tremendous impact on our understanding of many biological and pathological processes across different cell types, tissues and organisms, and even enable new approaches to drug development,” said Wyss Founding Director Donald Ingber, MD , Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and Professor of Bioengineering at Harvard John A. Paulson School of Engineering and Applied Sciences.
The study was also authored by Joseph Piccirilli, Ph.D., an expert in RNA chemistry and biochemistry and a professor at the University of Chicago. It was supported by the National Science Foundation (NSF; grant # CMMI-1333215, CCMI-1344915 and CBET-1729397), Air Force Office of Scientific Research (AFOSR; grant MURI FATE, # FA9550-15-1-0514 ), National Institutes of Health (NIH; grant # 5DP1GM133052, R01GM122797 and R01GM102489) and the Wyss Institute Molecular Robotics Initiative.