MSGID: 1:317/3 6270b055   
   PID: hpt/lnx 1.9.0-cur 2019-01-08   
   TID: hpt/lnx 1.9.0-cur 2019-01-08   
    Nanotechnology enables visualization of RNA structures at near-atomic   
   resolution    
      
     Date:   
         May 2, 2022   
     Source:   
         Wyss Institute for Biologically Inspired Engineering at Harvard   
     Summary:   
         Researchers have reported a fundamentally new approach to the   
         structural investigation of RNA molecules. ROCK, as it is called,   
         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. The team showed   
         that their method enables the structural analysis of the contained   
         RNA subunits with a technique known as cryo-electron microscopy   
         (cryo-EM).   
      
      
      
   FULL STORY   
   ==========================================================================   
   We live in a world made and run by RNA, the equally important sibling 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 modern day humans:   
   science has revealed that less than 3% of the human genome is transcribed   
   into messenger RNA (mRNA) molecules that in turn are translated into   
   proteins. In contrast, 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   
   needs to be deciphered at the level of its constituent atoms and   
   molecular bonds.   
      
   Researchers have routinely 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 effectiveness 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 the Wyss Institute for Biologically Inspired Engineering at Harvard   
   University, and Maofu Liao, Ph.D. at Harvard Medical School (HMS), has   
   reported a fundamentally new approach to the structural investigation   
   of RNA molecules.   
      
   ROCK, as it is called, 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 team showed that their method enables the structural analysis of the   
   contained RNA subunits with a technique known as cryo-electron microscopy   
   (cryo-EM). Their advance is reported in Nature Methods.   
      
   "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 Yin, who together with Liao led the study. "We expect   
   this advance to invigorate many areas of fundamental research and drug   
   development, including the burgeoning field of RNA therapeutics." Yin   
   also is a leader of the Wyss Institute's Molecular Robotics Initiative   
   and Professor in the Department of Systems Biology at HMS.   
      
   Gaining control over RNA Yin's team at the Wyss Institute has pioneered   
   various approaches that enable DNA and RNA molecules to self-assemble into   
   large structures based on different principles and requirements, including   
   DNA bricks and DNA origami. They hypothesized that such strategies could   
   also be used to assemble naturally occurring RNA molecules into highly   
   ordered circular complexes in which their freedom to flex and move is   
   highly restricted 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 a 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 Yin's group. "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."  Picturing   
   stabilized RNA In cryo-EM, many single particles are flash-frozen   
   at cryogenic temperatures to prevent any further movements, and then   
   visualized with an electron microscope and the help of computational   
   algorithms that compare the various aspects of a particle's 2D surface   
   projections and reconstruct its 3D architecture. Peng and Liu teamed   
   up with Liao and his former graduate student Franc,ois The'lot, Ph.D.,   
   the other co-first author of the study. Liao with his group has made   
   important contributions to the rapidly advancing cryo-EM field and the   
   experimental and computational analysis of single particles formed by   
   specific proteins.   
      
   "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 Liao,   
   who also is Associate Professor of Cell Biology at HMS. "Our approach   
   has opened the door to rapid structure determination of many RNAs by   
   cryo-EM." The integration of RNA nanotechnology and cryo-EM approaches   
   led the team to name their method "RNA oligomerization- enabled cryo-EM   
   via installing kissing loops" (ROCK).   
      
   To provide proof-of-principle for ROCK, the team focused 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   
   sequences of freshly-transcribed RNAs and have to be "spliced" out in   
   order for the mature RNA to be generated. 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   
   The'lot. "The RNA's core is resolved at 2.85 AA [one AAngstro"m 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 gotten there without ROCK   
   -- or at least not without considerably more resources."  Cryo-EM also   
   is able to capture molecules in different states if they, for example,   
   change their 3D conformation as part of their function. 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.   
      
   "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 occurring RNA molecules could have tremendous impact on   
   our understanding of many biological and pathological processes across   
   different cell types, tissues, and organisms, and even enable new drug   
   development approaches," said 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 Children's Hospital, and Professor of   
   Bioengineering at the 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 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's Molecular Robotics Initiative.   
      
      
   ==========================================================================   
   Story Source: Materials provided   
   by Wyss_Institute_for_Biologically_Inspired_Engineering_at   
   Harvard. Original written by Benjamin Boettner. Note: Content may be   
   edited for style and length.   
      
      
   ==========================================================================   
   Journal Reference:   
      1. Di Liu, Franc,ois A. The'lot, Joseph A. Piccirilli, Maofu Liao,   
      Peng Yin.   
      
         Sub-3-AA cryo-EM structure of RNA enabled by engineered   
         homomeric self- assembly. Nature Methods, 2022; DOI:   
         10.1038/s41592-022-01455-w   
   ==========================================================================   
      
   Link to news story:   
   https://www.sciencedaily.com/releases/2022/05/220502120510.htm   
      
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