Tag Archives: Peng Yin

Visualization of RNA structures at near-atomic resolution enabled by nanotechnology

The illustration that accompanies the research is both fascinating and baffling as its caption,

Caption: This illustration is inspired by the Paleolithic rock painting in the Lascaux cave, signifying the acronym of our method, ROCK. Figuratively, the patterns of the rock art in the background (brown) are the 2D projections 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 allowed the collaborators to build an atomic model of the RNA. Credit: Wyss Institute at Harvard University

This May 2, 2022 news item on ScienceDaily announces the research into RNA molecules made possible by ROCK (the technology being illustrated in the above),

We live in a world made and run by RNA [ribonucleic acid], 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.

A May 2, 2022 Wyss Institute for Biologically Inspired Engineering at Harvard University news release (also on EurekAlert) by Benjamin Boettner, which originated the news item, delves further into the imaging technology, Note: Links have been removed,

“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., 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.

Here’s a link to and a citation for the paper,

Sub-3-Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly by Di Liu, François A. Thélot, Joseph A. Piccirilli, Maofu Liao & Peng Yin. Nature Methods (2022) DOI: https://doi.org/10.1038/s41592-022-01455-w Published: 02 May 2022

This paper is behind a paywall.

Getting up to the size of a dust speck, the first ‘large’ self-assembling DNA crystals

An Oct. 19, 2014 news item on ScienceDaily describes the latest developments in ‘DNA nanotechnology’ research at the Wyss Institute for Biologically Inspired Engineering at Harvard University,

DNA has garnered attention for its potential as a programmable material platform that could spawn entire new and revolutionary nanodevices in computer science, microscopy, biology, and more. Researchers have been working to master the ability to coax DNA molecules to self assemble into the precise shapes and sizes needed in order to fully realize these nanotechnology dreams.

For the last 20 years, scientists have tried to design large DNA crystals with precisely prescribed depth and complex features — a design quest just fulfilled by a team at Harvard’s Wyss Institute for Biologically Inspired Engineering. The team built 32 DNA crystals with precisely-defined depth and an assortment of sophisticated three-dimensional (3D) features, an advance reported in Nature Chemistry.

It seems a bit of a misleading for the Wyss Institute to state the ‘team built’ the DNA crystals as they are self-assembling according to this Oct. 19, 2014 Wyss Institute news release (also on EurekAlert), which originated the news item,

The team used their “DNA-brick self-assembly” method, which was first unveiled in a 2012 Science publication when they created more than 100 3D complex nanostructures about the size of viruses. The newly-achieved periodic crystal structures are more than 1000 times larger than those discrete DNA brick structures, sizing up closer to a speck of dust, which is actually quite large in the world of DNA nanotechnology.

“We are very pleased that our DNA brick approach has solved this challenge,” said senior author and Wyss Institute Core Faculty member Peng Yin, Ph.D., who is also an Associate Professor of Systems Biology at Harvard Medical School, “and we were actually surprised by how well it works.”

The news release goes on to describe some of the issues with other self-assembly methods along with more details about the ‘DNA brick’ approach,

Scientists have struggled to crystallize complex 3D DNA nanostructures using more conventional self-assembly methods. The risk of error tends to increase with the complexity of the structural repeating units and the size of the DNA crystal to be assembled.

The DNA brick method uses short, synthetic strands of DNA that work like interlocking Lego® bricks to build complex structures. Structures are first designed using a computer model of a molecular cube, which becomes a master canvas. Each brick is added or removed independently from the 3D master canvas to arrive at the desired shape – and then the design is put into action: the DNA strands that would match up to achieve the desired structure are mixed together and self assemble to achieve the designed crystal structures.

“Therein lies the key distinguishing feature of our design strategy—its modularity,” said co-lead author Yonggang Ke, Ph.D., formerly a Wyss Institute Postdoctoral Fellow and now an assistant professor at the Georgia Institute of Technology and Emory University. “The ability to simply add or remove pieces from the master canvas makes it easy to create virtually any design.”

The modularity also makes it relatively easy to precisely define the crystal depth. “This is the first time anyone has demonstrated the ability to rationally design crystal depth with nanometer precision, up to 80 nm in this study,” Ke said. In contrast, previous two-dimensional DNA lattices are typically single-layer structures with only 2 nm depth.

“DNA crystals are attractive for nanotechnology applications because they are comprised of repeating structural units that provide an ideal template for scalable design features”, said co-lead author graduate student Luvena Ong.

Furthermore, as part of this study the team demonstrated the ability to position gold nanoparticles into prescribed 2D architectures less than two nanometers apart from each other along the crystal structure – a critical feature for future quantum devices and a significant technical advance for their scalable production, said co-lead author Wei Sun, Ph.D., Wyss Institute Postdoctoral Fellow.

“My preconceived notions of the limitations of DNA have been consistently shattered by our new advances in DNA nanotechnology,” said William Shih, Ph.D., who is co-author of the study and a Wyss Institute Founding Core Faculty member, as well as Associate Professor in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School and the Department of Cancer Biology at the Dana-Farber Cancer Institute. “DNA nanotechnology now makes it possible for us to assemble, in a programmable way, prescribed structures rivaling the complexity of many molecular machines we see in Nature.”

“Peng’s team is using the DNA-brick self-assembly method to build the foundation for the new landscape of DNA nanotechnology at an impressive pace,” said Wyss Institute Founding Director Don Ingber, M.D., Ph.D. “What have been mere visions of how the DNA molecule could be used to advance everything from the semiconductor industry to biophysics are fast becoming realities.”

Here’s a link to and a citation for the latest paper,

DNA brick crystals with prescribed depths by Yonggang Ke, Luvena L. Ong, Wei Sun, Jie Song, Mingdong Dong, William M. Shih, & Peng Yin. Nature Chemistry (2014) doi:10.1038/nchem.2083 Published online 19 October 2014

This paper is behind a paywall.