We live in a world made and ruled by RNA, the equally important sibling of the genetic molecule DNA. In fact, evolutionary biologists hypothesize that RNA existed and was self-replicating before the appearance of DNA and the proteins it encoded. Fast forward to modern humans: Science has shown that less than 3% of the human genome is transcribed into messenger RNA (mRNA) molecules, which in turn are translated into proteins. In contrast, 82% of these are transcribed into RNA molecules with other functions, many of which remain enigmatic.
To understand what a single RNA molecule does, its 3D structure must 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 studied with an X-ray (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 has begun, 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 on a fundamentally new approach to studying the structure of RNA molecules. ROCK, as it is known, uses an RNA nanotechnology technique that allows multiple identical RNA molecules to be assembled into a highly organized structure, significantly reducing the flexibility of individual RNA molecules and multiplying their molecular weight. Applied to known model RNAs with different sizes and functions as benchmarks, the team showed that their method allows structural analysis of the contained RNA subunits using a technique known as cryo-electron microscopy (cryo-EM). Your advance will be reported in natural methods.
“ROCK breaks through the current limitations of RNA structure studies and enables the decoding of 3D structures of RNA molecules that are difficult or impossible to access with existing methods, at near-atomic resolution,” said Yin, who co-authored with Liao led the study. “We expect this advance will revitalize many areas of basic research and drug development, including the burgeoning area of RNA therapeutics.” Yin is also director of the Wyss Institute’s Molecular Robotics Initiative and Professor in the Department of Systems Biology at HMS.
Gain control over RNA
Yin’s team at the Wyss Institute has developed various approaches that allow DNA and RNA molecules to self-assemble into large structures based on different principles and requirements, including DNA building blocks 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 of movement is severely restricted by specifically linking them together. Many RNAs fold in complex but predictable ways, with small segments base pairing with each other. The result is often a stabilized “core” and “stem loops” that bulge out into 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 that an overall stabilized ring containing multiple copies of the RNA of interest can be formed,” said Di Liu, Ph.D., one of two first authors and 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 initial success.”
Imaging stabilized RNA
In cryo-EM, many individual particles are snap frozen at cryogenic temperatures to prevent further movement and then visualized using an electron microscope and using computer algorithms that compare the different 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 François Thélot, Ph.D., the study’s other co-first author. Liao and his group have made important contributions to the rapidly developing field of cryo-EM and experimental and computational analysis of single particles composed of specific proteins.
“Cryo-EM has great advantages over traditional methods in visualizing high-resolution details of biological molecules, including proteins, DNAs and RNAs, but the small size and tendency to move of most RNAs prevents successful determination of RNA structures. Our novel method of assembling RNA multimers solves both of these problems simultaneously by increasing the size of RNA and decreasing its movement,” said Liao, who is also an associate professor of cell biology at HMS. “Our approach has the door to fast Structure determination of many RNAs opened up by cryo-EM.” The integration of RNA nanotechnology and cryo-EM approaches prompted the team to name their method “RNA-Oligomerizable Cryo-EM via Installation of Kissing Loops” (ROCK).
To provide proof-of-principle for ROCK, the team focused on a large intron RNA Tetrahymenaa unicellular organism, and a small intron RNA azoarcus, a nitrogen-fixing bacterium, and the so-called FMN riboswitch. Intron RNAs are non-coding RNA sequences that are scattered throughout the sequences of freshly transcribed RNAs and must 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. When it binds one of them, flavin mononucleotide (FMN), it changes its 3D conformation and suppresses the synthesis of its mother RNA.
“The assembly of Tetrahymena Group I intron into a ring-shaped structure made the samples more homogeneous and enabled the use of computational tools that exploit the symmetry of the assembled structure. While our data set is relatively modest, the inherent advantages of ROCK allowed us to resolve the structure with unprecedented resolution,” Thélot said. “The core of the RNA is resolved at 2.85 Å [one Ångström is one ten-billions (US) of a meter and the preferred metric used by structural biologists], which reveals detailed features of the nucleotide bases and the sugar backbone. I don’t think we could have gotten there without ROCK – or at least not without significantly more resources.”
Cryo-EM is also able to detect molecules in different states, for example when they change their 3D conformation as part of their function. Applying ROCK to the azoarcus Intron RNA and the FMN riboswitch, the team was able to identify the different conformations that the azoarcus Intron transitions 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 demonstrates how RNA nanotechnology can act as an accelerator to advance other disciplines. The ability 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 Donald Ingber, founding director of Wyss, 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’s 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.