La nanotecnología permite la visualización en 3D de estructuras de ARN cruciales con una resolución casi atómica

Esta ilustración está inspirada en la pintura rupestre paleolítica de la cueva de Lascaux, que significa el acrónimo de nuestro método, ROCK. En sentido figurado, los patrones del arte rupestre en el fondo (marrón) son las proyecciones 2D de la construcción dimérica diseñada del intrón del grupo I de Tetrahymena, mientras que el objeto principal en el frente (azul) es el mapa crio-EM 3D reconstruido del dímero, con un monómero en foco y refinado a la alta resolución que permitió a los colaboradores construir un modelo atómico del ARN. Crédito: Instituto Wyss de la Universidad de Harvard

combinación de nucleico[{” attribute=””>acid nanotechnology and cryo-EM gives unprecedented insights into the structures of large and small RNAs, advancing

Ribonucleic acid (RNA) is a polymeric molecule that is essential in various biological roles in coding, decoding, regulation and expression of genes. Both RNA and deoxyribonucleic acid (DNA) are nucleic acids. Along with lipids, proteins, and carbohydrates, nucleic acids constitute one of the four major macromolecules essential for all known forms of life. RNA, like DNA, is assembled as a chain of nucleotides, but unlike DNA, RNA is found in nature as a single strand folded onto itself, rather than a paired double strand.

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 the journal 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.

“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. We expect this advance to invigorate many areas of fundamental research and drug development, including the burgeoning field of RNA therapeutics.”

Peng Yin

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.

ROCK RNA

In ROCK (RNA oligomerization-enabled cryo-EM via installing kissing loops), a target RNA is engineered for the self-assembly of a closed homomeric ring via kissing-loop sequences (red) that are installed onto functionally nonessential, peripheral helices (blue). After identifying engineerable nonessential peripheral helices, the length of the helices connecting the kissing-loop motif and the core of the target RNA is computationally optimized. An RNA construct with multiple individual subunits of the target RNA is transcribed, assembled, and then purified by gel electrophoresis, before it can be analyzed via the cryo-EM method. Credit: Wyss Institute at Harvard University

“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 an 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.

ROCK-Enabled Structure

In their analysis of the Tetrahymena group I intron, the researchers collected about 105,000 single-particle cryo-EM images of the ROCK-enabled structure, and over a series of computational analysis steps reconstructed its structure, reaching an overall resolution of 2.98 Å, and a resolution of 2.85 Å for the core of the structure. The final models provided a detailed view of the Tetrahymena group I intron, including the previously unknown peripheral domains (shown in brown and purple), which constitute a belt surrounding the core. Credit: Wyss Institute at Harvard University

“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], revelando características detalladas de las bases de nucleótidos y la columna vertebral de azúcar. No creo que hubiéramos podido llegar allí sin ROCK, o al menos no sin una cantidad considerablemente mayor de recursos”.

Cryo-EM también puede capturar moléculas en diferentes estados si, por ejemplo, cambian su conformación 3D como parte de su función. Aplicando ROCK al Azoarco intrón ARN y el ribointerruptor FMN, el equipo logró identificar las diferentes conformaciones que el Azoarco transiciones de intrones durante su proceso de auto-empalme, y para revelar la rigidez conformacional relativa del sitio de unión al ligando del riboconmutador FMN.

“Este estudio de Peng Yin y sus colaboradores muestra con elegancia cómo la nanotecnología de ARN puede funcionar como un acelerador para el avance de otras disciplinas. Ser capaz de visualizar y comprender las estructuras de muchas moléculas de ARN naturales podría tener un tremendo impacto en nuestra comprensión de muchos procesos biológicos y patológicos en diferentes tipos de células, tejidos y organismos, e incluso permitir nuevos enfoques de desarrollo de fármacos”, dijo el director fundador de Wyss. Donald Ingber, MD, Ph.D., quien también es el Judah Folkman Profesor de Biología Vascular en la Escuela de Medicina de Harvard y el Hospital de Niños de Boston, y el Hansjörg Wyss Profesor de Ingeniería Bioinspirada en la Escuela de Ingeniería y Ciencias Aplicadas John A. Paulson de Harvard.

Referencia: “Estructura sub-3-Å crio-EM del ARN habilitada por autoensamblaje homomérico diseñado” por Di Liu, François A. Thélot, Joseph A. Piccirilli, Maofu Liao y Peng Yin, 2 de mayo de 2022, Métodos de la naturaleza.
DOI: 10.1038/s41592-022-01455-w

El estudio también fue escrito por Joseph Piccirilli, Ph.D., experto en química y bioquímica del ARN y profesor de The[{” attribute=””>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.

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