Ribonucleic acids (RNA) play a crucial role in the biological functions of living organisms. The cellular functions and properties of RNA rely significantly on various chemical modifications. Understanding and evaluating these modifications are essential for delving into RNA biology at molecular and mechanistic levels. The chemical synthesis of modified RNA involves intricate processes to incorporate specific alterations into RNA molecules. Recent advancements in the field of chemical RNA synthesis have paved the way for controlled modifications of RNA sequences, both in vitro and in vivo which include [1]:
Solid-Phase Synthesis of Modified RNA
One of the key techniques in the field of chemical RNA synthesis is solid-phase RNA synthesis. This method enables stepwise chemical assembly of RNA molecules, starting from anchored RNA sequences on solid supports. Solid-phase RNA synthesis involves the stepwise assembly of RNA molecules on a solid support, typically using phosphoramidite chemistry. The RNA building blocks are added one at a time in a sequential manner, starting from the 3' end of the RNA molecule. Each synthesis cycle consists of deprotecting the 5' end of the RNA fragment and adding the next nucleotide, leading to the gradual elongation of the RNA chain. This process continues until the desired RNA sequence, with specific modifications, is achieved.
Enzymatic Ligation of Chemically Modified RNAs
Enzymatic ligation of synthetic RNA sequences serves as a powerful tool for producing RNA molecules with multiple site-specific modifications. This technique is instrumental in creating RNA molecules with complex structures, such as those required for investigating the effects of individual modifications within the context of RNA functions. Enzymatic ligation of chemically modified RNAs involves the use of commercial T4 DNA ligase, along with a complementary DNA splint of approximately 20 to 25 nucleotides in length. The ligation process relies on the presence of a 5'-phosphate on the RNA fragment to be ligated to the 3' end of another RNA fragment. This phosphate group can be incorporated directly during the final coupling cycle of RNA solid-phase synthesis, simplifying the workflow and eliminating the need for separate enzymatic phosphorylation steps.
Cofactor Utilizing Ribozymes for RNA Modification
Another innovative approach for modifying RNA involves the use of ribozymes that utilize cofactors to transfer functional groups onto RNA molecules. These ribozymes contain recognition arms that hybridize to specific RNA target sequences, allowing for the precise modification of nucleosides within RNA molecules. By leveraging engineered ribozymes in a protein enzyme-free system, researchers can achieve site-specific in vivo RNA labeling, offering a promising avenue for studying RNA modifications in a controlled manner.
The chemical synthesis of modified RNA has seen significant advancements in recent years, enabling researchers to tailor RNA molecules with specific modifications for various biological and biotechnological applications. By harnessing the power of solid-phase synthesis, enzymatic ligation, and ribozyme-based modification strategies, scientists can explore the intricate world of RNA biology with enhanced precision and control. Our company is a leading supplier of nucleosides / nucleotides synthesis services. Contact us to learn more about how we can support your scientific endeavors and help you achieve your goals.
Reference
- Flemmich L, Bereiter R, Micura R. Chemical Synthesis of Modified RNA. Angew Chem Int Ed Engl. 2024 Mar 26:e202403063.
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