This illustration is influenced by the Paleolithic rock painting in the Lascaux cavern, symbolizing the acronym of our technique, ROCK. Figuratively, the patterns of the rock art in the background (brown) are the 2D forecasts of the engineered dimeric construct of the Tetrahymena group I intron, while the main object in the front (blue) is the reconstructed 3D cryo-EM map of the dimer, with one monomer in focus and refined to the high resolution that permitted the collaborators to construct an atomic model of the RNA. Credit: Wyss Institute at Harvard University
Mix of nucleic acid nanotechnology and cryo-EM gives unprecedented insights into the structures of little and big RNAs, advancing RNA biology and drug design.
We live in a world created and run by RNA, the similarly crucial sibling of the genetic particle DNA. In contrast, 82% of it is transcribed into RNA molecules with other functions many of which are yet unidentified.
Ribonucleic acid (RNA) is a polymeric molecule that is essential in numerous biological roles in coding, translating, policy and expression of genes. RNA, like DNA, is assembled as a chain of nucleotides, however unlike DNA, RNA is found in nature as a single strand folded onto itself, rather than a paired double strand.
To comprehend what an individual RNA molecule does, its 3D structure needs to be figured out at the level of its constituent atoms and molecular bonds. Scientists have actually consistently studied DNA and protein molecules by turning them into regularly jam-packed crystals that can be taken a look at with an X-ray beam (X-ray crystallography) or radio waves (nuclear magnetic resonance). Nevertheless, these techniques can not be applied to RNA molecules with nearly the very same efficiency due to the fact that their molecular structure and structural versatility prevent them from easily forming crystals.
Now, a research study cooperation led by Wyss Core Faculty member Peng Yin, Ph.D. at the Wyss Institute for Biologically Inspired Engineering at Harvard University, and Maofu Liao, Ph.D. at Harvard Medical School (HMS), has reported an essentially new approach to the structural examination of RNA particles. ROCK, as it is called, uses an RNA nanotechnological technique that allows it to put together numerous similar RNA particles into an extremely organized structure, which substantially decreases the versatility of specific RNA molecules and multiplies their molecular weight. Applied to widely known design RNAs with different sizes and functions as benchmarks, the team showed that their method enables the structural analysis of the consisted of RNA subunits with a method understood as cryo-electron microscopy (cryo-EM). Their advance is reported in the journal Nature Methods.
” ROCK is breaking the current limits of RNA structural examinations and makes it possible for 3D structures of RNA molecules to be opened that are impossible or difficult to access with existing approaches, and at near-atomic resolution,” stated Yin, who together with Liao led the study. “We anticipate this advance to rejuvenate numerous locations of basic research and drug advancement, consisting of the blossoming field of RNA therapeutics.” Yin likewise is a leader of the Wyss Institutes Molecular Robotics Initiative and Professor in the Department of Systems Biology at HMS.
” ROCK is breaking the current limits of RNA structural investigations and allows 3D structures of RNA particles to be opened that are tough or difficult to gain access to with existing approaches, and at near-atomic resolution. We anticipate this advance to rejuvenate many locations of basic research and drug development, including the burgeoning field of RNA therapeutics.”
— Peng Yin
Gaining control over RNA
Yins team at the Wyss Institute has actually pioneered numerous methods that make it possible for DNA and RNA particles to self-assemble into large structures based on different concepts and requirements, including DNA bricks and DNA origami. They hypothesized that such strategies might likewise be utilized to assemble naturally happening RNA molecules into extremely purchased circular complexes in which their liberty to flex and move is extremely limited by particularly connecting them together. Many RNAs fold in complex yet foreseeable ways, with small sectors base-pairing with each other. The result frequently is a supported “core” and “stem-loops” bulging out into the periphery.
In ROCK (RNA oligomerization-enabled cryo-EM by means of setting up kissing loops), a target RNA is engineered for the self-assembly of a closed homomeric ring through kissing-loop sequences (red) that are installed onto functionally unnecessary, peripheral helices (blue). An RNA construct with numerous private subunits of the target RNA is transcribed, assembled, and then cleansed by gel electrophoresis, prior to it can be examined through the cryo-EM technique.
” In our technique we set up kissing loops that link various peripheral stem-loops belonging to 2 copies of a similar RNA in such a way that permits an overall stabilized ring to be formed, containing multiple copies of the RNA of interest,” said Di Liu, Ph.D., one of two first-authors and a Postdoctoral Fellow in Yins group. “We hypothesized that these higher-order rings might be evaluated with high resolution by cryo-EM, which had actually been applied to RNA particles with very first success.”
Picturing stabilized RNA
In cryo-EM, numerous single particles are flash-frozen at cryogenic temperature levels to avoid any further motions, and after that envisioned with an electron microscopic lense and the assistance of computational algorithms that compare the numerous elements of a particles 2D surface area projections and reconstruct its 3D architecture. Peng and Liu partnered with Liao and his previous graduate student François Thélot, Ph.D., the other co-first author of the research study. Liao with his group has actually made essential contributions to the quickly advancing cryo-EM field and the experimental and computational analysis of single particles formed by specific proteins.
” Cryo-EM has fantastic advantages over traditional approaches in seeing high-resolution details of biological particles including proteins, rnas and dnas, however the small size and moving tendency of a lot of RNAs avoid successful decision of RNA structures. Our unique approach of putting together RNA multimers solves these two issues at the exact same time, by increasing the size of RNA and lowering its movement,” stated Liao, who also is an Associate Professor of Cell Biology at HMS. “Our approach has unlocked to rapid structure determination of numerous RNAs by cryo-EM.” The combination of RNA nanotechnology and cryo-EM techniques led the team to name their approach “RNA oligomerization-enabled cryo-EM by means of installing kissing loops” (ROCK).
To offer proof-of-principle for ROCK, the team concentrated on a large intron RNA from Tetrahymena, a single-celled organism, and a small intron RNA from Azoarcus, a nitrogen-fixing bacterium, as well as the so-called FMN riboswitch. Intron RNAs are non-coding RNA sequences scattered throughout the series of freshly-transcribed RNAs and have to be “spliced” out in order for the mature RNA to be generated. The FMN riboswitch is discovered in bacterial RNAs included in the biosynthesis of flavin metabolites stemmed from vitamin B2. Upon binding one of them, flavin mononucleotide (FMN), it changes its 3D conformation and reduces the synthesis of its mother RNA.
In their analysis of the Tetrahymena group I intron, the researchers gathered about 105,000 single-particle cryo-EM images of the ROCK-enabled structure, and over a series of computational analysis actions rebuilded its structure, reaching a general resolution of 2.98 Å, and a resolution of 2.85 Å for the core of the structure. The final models offered an in-depth view of the Tetrahymena group I intron, consisting of the previously unknown peripheral domains (displayed in brown and purple), which make up a belt surrounding the core. Credit: Wyss Institute at Harvard University
” The assembly of the Tetrahymena group I intron into a ring-like structure made the samples more homogenous, and made it possible for the use of computational tools leveraging the proportion of the assembled structure. While our dataset is reasonably modest in size, ROCKs natural advantages permitted us to solve the structure at an unmatched resolution,” stated Thélot. I do not think we could have gotten there without ROCK– or at least not without significantly more resources.”
Cryo-EM also has the ability to catch molecules in various states if they, for example, alter their 3D conformation as part of their function. Using ROCK to the Azoarcus intron RNA and the FMN riboswitch, the team handled to recognize the various conformations that the Azoarcus intron shifts through throughout its self-splicing procedure, and to reveal the relative conformational rigidness of the ligand-binding website of the FMN riboswitch.
” This study by Peng Yin and his collaborators elegantly shows how RNA nanotechnology can work as an accelerator to advance other disciplines. Being able to visualize and understand the structures of many naturally taking place RNA molecules might have significant effect on our understanding of many biological and pathological processes across different cell organisms, tissues, and types, and even allow new drug advancement techniques,” stated Wyss Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Childrens Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.
Reference: “Sub-3-Å cryo-EM structure of RNA made it possible for by engineered homomeric self-assembly” by Di Liu, François A. Thélot, Joseph A. Piccirilli, Maofu Liao and Peng Yin, 2 May 2022, Nature Methods.DOI: 10.1038/ s41592-022-01455-w.
The study was also authored by Joseph Piccirilli, Ph.D., a professional in RNA chemistry and biochemistry and Professor at The University of Chicago. It was supported by the National Science Foundation (NSF; grant # CBET-1729397, cmmi-1333215, and ccmi-1344915), Air Force Office of Scientific Research (AFOSR; grant MURI FATE, #FA 9550-15-1-0514), National Institutes of Health (NIH; grant # 5DP1GM133052, R01GM122797, and R01GM102489), and the Wyss Institutes Molecular Robotics Initiative.
ROCK, as it is called, utilizes an RNA nanotechnological technique that enables it to assemble several identical RNA particles into an extremely organized structure, which substantially decreases the versatility of individual RNA molecules and multiplies their molecular weight.” ROCK is breaking the present limitations of RNA structural investigations and allows 3D structures of RNA particles to be opened that are impossible or hard to gain access to with existing techniques, and at near-atomic resolution,” said Yin, who together with Liao led the research study.” Cryo-EM has terrific advantages over conventional techniques in seeing high-resolution details of biological molecules including rnas, dnas and proteins, but the small size and moving tendency of many RNAs prevent successful decision of RNA structures. The combination of RNA nanotechnology and cryo-EM techniques led the group to call their technique “RNA oligomerization-enabled cryo-EM via installing kissing loops” (ROCK).
Intron RNAs are non-coding RNA series spread throughout the sequences of freshly-transcribed RNAs and have to be “entwined” out in order for the fully grown RNA to be created.