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.
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 François Thélot, 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 Thélot. “The RNA’s 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 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
News
Common Medication Could Save Half a Million Lives Each Year – So Why Isn’t It?
A recent study conducted by scientists at the University of Southern California sheds light on the reasons why children are not receiving an affordable and effective diarrhea treatment. Medical professionals in developing nations are [...]
X Marks the Spot: AI’s Treasure Maps Lead to Early Disease Detection
Medical diagnostics expert, doctor’s assistant, and cartographer are all fair titles for an artificial intelligence model developed by researchers at the Beckman Institute for Advanced Science and Technology. Their new model accurately identifies tumors [...]
Scientists Discover Method To Identify Alzheimer’s Disease Before It Progresses to Dementia
Researchers at Aarhus University have discovered a method to identify Alzheimer’s disease before it progresses to dementia, potentially opening up new avenues for treatment. A groundbreaking study could pave the way for early detection [...]
Startling Discovery: COVID-19 Virus Can Stay in the Body More Than a Year After Infection
The COVID-19 virus can persist in the blood and tissue of patients for more than a year after the acute phase of the illness has ended, according to new research from UC San Francisco that offers potential [...]
New bioengineered protein design shows promise in fighting COVID-19
In the wake of the COVID-19 pandemic, scientists have been racing to develop effective treatments and preventatives against the virus. A recent scientific breakthrough has emerged from the work of researchers aiming to combat [...]
Sugar-coated gold nanoparticles can quickly eliminate bacterial infections, no antibiotics required
If left to their own devices, bacteria on our teeth or wounded skin can encase themselves in a slimy scaffolding, turning into what is called biofilm. These bacteria wreak havoc on our tissue and, [...]
Liquid Lightning: Nanotechnology Unlocks New Energy
EPFL researchers have discovered that nanoscale devices harnessing the hydroelectric effect can harvest electricity from the evaporation of fluids with higher ion concentrations than purified water, revealing a vast untapped energy potential. Evaporation is a natural [...]
Unmasking the Illusion: AI-Generated Faces Challenge Perceptions
Research shows survey participants duped by AI-generated images nearly 40 percent of the time. If you recently had trouble figuring out if an image of a person is real or generated through artificial intelligence [...]
New Discovery Reveals How Cells Defend Themselves During Stressful Situations
Stress granules play a crucial role in the stress response, arising from the aggregation of non-translating mRNAs and proteins. Although significant knowledge exists about stress granules, the mechanisms behind their mRNA localization remain partially [...]
Scientists use a new type of nanoparticle that can both deliver vaccines and act as an adjuvant
Many vaccines, including vaccines for hepatitis B and whooping cough, consist of fragments of viral or bacterial proteins. These vaccines often include other molecules called adjuvants, which help to boost the immune system's response [...]
Not Science Fiction: How Optical Neural Networks Are Revolutionizing AI
A novel architecture for optical neural networks utilizes wavefront shaping to precisely manipulate the travel of ultrashort pulses through multimode fibers, enabling nonlinear optical computation. Present-day artificial intelligence systems rely on billions of adjustable [...]
Turning skin cells into limb cells sets the stage for regenerative therapy
In a collaborative study, researchers from Kyushu University and Harvard Medical School have identified proteins that can turn or “reprogram” fibroblasts — the most commonly found cells in skin and connective tissue — into [...]
AI reveals prostate cancer is not just one disease
Artificial Intelligence has helped scientists reveal a new form of aggressive prostate cancer, which could revolutionise how the disease is diagnosed and treated in the future. A Cancer Research UK-funded study, published in Cell Genomics, has revealed [...]
New Study Finds That Persistent COVID-19 Infections Are Surprisingly Common
Recent research conducted by the University of Oxford has found that a high proportion of SARS-CoV-2 infections in the general population lead to persistent infections lasting a month or more. The findings have been published in the journal Nature. [...]
Innovative nanosheet method revolutionizes brain imaging for multi-scale and long-term studies
The human brain has billions of neurons. Working together, they enable higher-order brain functions such as cognition and complex behaviors. To study these higher-order brain functions, it is important to understand how neural activity [...]
Scientists Have Discovered a Potential Universal Antivenom
Scientists at Scripps Research identified antibodies that protect against a host of lethal snake venoms. Scripps Research scientists have developed an antibody that can block the effects of lethal toxins in the venoms of [...]