The combination of nucleic acid nanotechnology and cryo-EM provides incredible insight into the structures of large and small RNAs (RNA structures in almost atomic resolution), enabling major advances in RNA biology and drug design.
We live in a world created and driven by RNA, the same important sister of the genetic DNA molecule. In fact, evolutionary biologists assume that RNA exists and replicates before DNA is discovered. Fast forward to modern humans: Science has revealed that less than 3% of the human genome is transcribed into messenger RNA (mRNA) molecules, which translate into egg whites. In contrast, 82% of this is transcribed into RNA molecules (RNA structures in almost atomic resolution) with other functions, many of which remain mysterious.
To understand what a single RNA molecule does, its 3D structure must be determined at the level of its atomic components and molecular bonds. Researchers regularly study DNA and egg white molecules by turning them into regularly packed crystals that can be examined by X-rays (X-ray crystallography) or radio waves (nuclear magnetic resonance). However, these methods do not apply to RNA molecules with almost identical efficiency, as their molecular composition and structural flexibility prevent them from easily forming crystals. Today, a research collaboration led by Wyss Primary School member Peng Yin, Ph.D. from the Wyss Institute for Biologically Inspired Engineering at Harvard University and Maofu Liao, Ph.D. at Harvard Medical School (HMS), introduces a fundamentally new approach to the structural research of RNA molecules (RNA structures in almost atomic resolution). ROCK, as it is called, uses an RNA nanotechnology technique that allows it to assemble many identical RNA molecules into a highly organized structure, which reduces the flexibility of individual cells. RNA molecules and increase their molecular weight. Using known RNA models with different sizes and functions as benchmarks, the team showed that their method can perform structural analysis of RNA subunits using a technique known as cryo-electron microscopy (cryo-EM). Their progress is reported in Nature Methods.
“ROCK breaks the current limitations of RNA structural research and allows you to unlock 3D structures of RNA molecules that are difficult or impossible to achieve with current methods and near atomic resolution,” said Yin, who led the study with Liao. “We anticipate that these advances will stimulate many aspects of basic research and development in medicine, including the evolving field of RNA therapeutics.” Yin is also the head of the Wyss Institute’s Molecular Robotics Initiative and a professor in the Department of Systems Biology at HMS.
Gain control of RNA
The Yin team at the Wyss Institute has pioneered a number of methods that allow DNA and RNA molecules to assemble into large structures based on various principles and requirements, including DNA cubes and origami DNA. They confirmed that such strategies can also be used to collect natural RNA molecules in highly ordered circular complexes, where their freedom of bending and movement is severely limited by their specific binding. . Many RNAs are composed in a complex but predictable way, with small parts joined together. The result is usually a strong “core” and “stem loops” that bulge on the periphery. “In our method, we have installed ‘kissing loops’ that connect several peripheral stem loops that belong to two copies of the same RNA in a way that allows for the creation of a generally strong ring with many copies of the RNA of interest,” he said. Di Liu, Ph.D., one of the first two authors and a postdoctoral fellow of the Yin group. “We hypothesize that these higher order rings can be analyzed in high resolution using cryo-EM, applied to RNA molecules with initial success.”
Characterization of stabilized RNA
In cryo-EM, many particles are frozen at cryogenic temperatures to prevent further movement, and then observed under an electron microscope using computational algorithms that compare various aspects of 2D particle surface projections and reconstruct their 3D architecture. . Peng and Liu collaborated with Lia and his former graduate student François Thélot, Ph.D., another co-author of the study. Liao and his team contributed significantly to the rapid development of the cryo-EM field and the experimental and computational analysis of particles formed from specific egg proteins.
Cryo-EM has many advantages over traditional methods of viewing details using high-resolution biological molecules, including proteins, DNA, and RNA, but the small size and motility potential of most RNA prevents successful determination of RNA structures. Our new method of assembling RNA multimers solves these two problems simultaneously by increasing RNA size and reducing mobility, “said Liao, who is also an associate professor of cell biology at HMS.” Our method opens the door to a combination of RNA nanotechnology, and the cryo-EM methods lead the team to call its method “cryo-EM with the support of RNA oligomerization by the installation of clumps” (ROCK).
To provide evidence of the ROCK principle, the team focused on large intron RNA from Tetrahymena, a single-celled organism, and small intron RNA from Azoarcus, a nitrogen-fixing bacterium, as well as the so-called FMN riboswitch. . Intron RNAs are non-coding RNA sequences that are scattered over the sequence of newly transcribed RNAs and must be “cleaved” to form adult RNA. FMN riboswitch is found in bacterial RNAs involved in the biosynthesis of vitamin B2-derived flavin metabolites. By binding to one of them, flavin mononucleotide (FMN), it changes its 3D conformation and inhibits the synthesis of its parent RNA.
“The composition of the Tetrahymena I intron in the ring structure makes the samples more homogeneous and allows the use of computational tools that use composite structure symmetry. While our dataset is relatively modest in size, the inherent benefits of ROCK allow us to solve the structure with unprecedented resolution,” said Thélot. “The RNA core is resolved at 2.85 Å [Angstrom ten billion (US) per meter and the preferred metric” used by structural biologists], revealing detailed characteristics. nucleotide bases and the sugar backbone. I don’t think we could get there without ROCK – or at least not without a lot of resources. ”
Cryo-EM is also able to capture molecules in different states because, for example, it changes its 3D conformation as part of its function. By applying ROCK to the Azoarcus intron RNA and the FMN riboswitch, the team was able to identify the different conformations that the Azoarcus intron undergoes during the self-cleavage process and to detect the relative conformational rigidity of the ligand binding site on the FMN riboswitch. . .
