Cryo-EM Analysis of High-resolution RNA Structures

RNA is ubiquitous in animals, plants, microorganisms, and some viruses and bacteriophages, and RNA and protein biosynthesis are closely related. RNA can act as a "messenger" to convey the genetic information on DNA and use it for protein synthesis. As a biological macromolecule, RNA has all the biological functions of DNA and protein in life activities and plays the same critical role as protein. Analyzing their three-dimensional structure is also a problem that scientists have been exploring for a long time. However, the analysis of the three-dimensional structure of RNA has always been limited by factors such as the small molecular weight and high flexibility of RNA. It is challenging to achieve whether it relies on cryo-electron microscopy or other structural analysis methods. In response to this urgent problem, Dr. Maofu Liao and Dr. Yin Peng from Harvard University collaborated to use ROCK (Cryo-EM analysis of high-resolution RNA structures) technology to transform RNA, empower cryo-electron microscopy, and analyze the high-resolution structures of various RNAs. It further expands the application scenarios of cryo-electron microscopy. Also, it opens a new situation for revealing the life activities involved in RNA and the development of drugs around RNA.

Figure 1. Kissing-Loop installation model.

By constructing a kissing-loop, ROCK technology can connect different peripheral Kissing-loops of two copies of the same RNA to form an overall stable loop that contains multiple copies of the target RNA. The RNA of interest is designed to self-assemble into a closed homologous loop through kissing hairpin sequences positioned on peripheral helices that are not essential for function. After identifying the editable non-essential peripheral helices, the length of the helix connecting the kissing hairpin motif to the RNA core of interest is computationally optimized. RNA constructs with multiple individual subunits of the RNA of interest are transcribed, assembled, purified by gel electrophoresis, and structurally elucidated by cryo-EM.

Figure 2. The workflow of ROCK.

Compared with traditional methods, cryo-EM has excellent advantages in resolving high-resolution structural details of biomolecules, including proteins, DNA, and RNA, but the small molecular weight and high flexibility of most RNAs make their structures difficult to resolve. The RNA polymer assembled by ROCK technology solves these two problems at the same time. By increasing the molecular weight of RNA and reducing its flexibility, our method opens the door to the field of RNA structure analysis based on cryo-electron microscopy. Visualizing the molecular structure of RNA in its native state greatly impacts the understanding of biological and pathological processes in different cell types, tissues, and organisms, and even enables new approaches to drug development.

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Reference

1. Liu D., et al. Sub-3-Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly. Nature Methods. 2022, 19(5): 576-585.