Protein-free RNA Structure Determination

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RNA is a versatile molecule that can perform a wide range of functions within the cell, often without needing to bind to a protein. These functions include information transfer, catalysis, gene regulation, structural roles, and more, with particular therapeutic attention paid to catalysis and gene regulation. Solving the structure of such RNAs is integral to understanding their diverse functions in cellular processes. 

RNA molecules showing enzymatic activity are categorized as ribozymes. Similar to proteins, ribozymes possess catalytic capabilities, challenging the conventional notion that only proteins could serve as enzymes. Ribozymes can be classified in several structural classes, including self-splicing, hammerhead, hairpin, group I and group II introns, each one with specific catalytic functions.

The Potential of Ribozyme Therapy and RNA Molecules

In recent years, researchers have explored the potential of ribozymes for therapeutic applications by using synthetic ribozymes to target and modify specific RNA sequences, including those within viral RNA or disease-associated mRNA. This approach, known as ribozyme therapy, has incredible potential in treating a wide range of diseases, with applications in oncology, genetic and neurological disorders, and inflammatory diseases. While it is in the early stages of development, continuing research into RNA engineering and delivery systems is expected to enhance the efficacy and versatility of ribozyme therapy. However, the dynamic nature of RNA structure has made it a difficult molecule to study. Cryo-EM can help.

Unlock the Potential of RNA with Protein-free Cryo-EM Structure Determination

To date, X-ray crystallography (XRC) and nuclear magnetic resonance (NMR) have been the main techniques for structural biologists looking to solve the structures of biomolecules. However, these methods often fail when it comes to elucidating the structure of RNA molecules. The inherent heterogeneity of RNA, caused by the flexible ribose and phosphate backbone, the weak long-range tertiary interactions, the existence of alternative conformations, and the dynamic nature of RNA in multiple functional states makes obtaining  RNA crystals difficult. On the other hand, while NMR can be used for solution structure and access conformational dynamics, it is limited to relatively small RNAs (< 100 nts) or smaller regions within larger RNAs.

Cryo-EM allows researchers to view biological molecules at near atomic resolution, bypassing the need for crystallization, and it doesn’t have an upper size limit, thus negating the challenges that make XRC and NMR impractical for elucidating RNA molecules. Using cryo-EM it is possible to image and classify individual RNA particles, allowing researchers to simultaneously examine the structure of RNA, as well as interrogate its conformational dynamics. More importantly, cryo-EM can visualize RNA molecules in complex with proteins, but also in its native, unbound state, without the influence of associated protein.  Recent technological and experimental advances  are allowing smaller and more flexible structures to be solved, thus providing the possibility of analyze in details RNA molecules that were until a few years ago beyond reach.

Advances in Cryo-EM have  greatly improved the ability to study and elucidate the structure of RNA molecules, allowing researchers unprecedented insights into the functions and mechanisms of RNA. These insights can contribute to exciting breakthroughs in the development of RNA-based therapies and novel drug targets for new and innovative therapeutic interventions. Learn more about their potential in our blog "Unlocking the Secrets of RNA: Protein-Free Structure Determination with Cryo-Electron Microscopy" here.

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