Introduction
Ribonucleic acid (RNA) plays a pivotal role in various biological processes, including protein synthesis, gene regulation, and cellular signaling. The discovery and understanding of diverse RNA functions have propelled significant advancements in biomedical research and therapeutics. Central to these advancements is the ability to chemically modify RNA molecules to enhance their stability, functionality, and interaction with other biomolecules.
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Chemical modifications of RNA have enabled the development of novel therapeutic strategies, such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), which can modulate gene expression with high specificity. Additionally, these modifications have provided powerful tools for studying RNA biology, allowing researchers to probe RNA structures, dynamics, and interactions with unprecedented precision.
Fig. 1 Sites of common RNA modifications (Röthlisberger P., et al. 2019).
Modifications of the Phosphodiester Backbone
The phosphodiester backbone of RNA, which links nucleotides together, is a critical target for chemical modifications aimed at enhancing RNA stability and function. Modifications at this site can improve resistance to nucleases, alter hybridization properties, and facilitate the introduction of functional groups for further chemical transformations.
One of the most extensively studied backbone modifications involves the replacement of a non-bridging oxygen atom in the phosphodiester linkage with a sulfur atom, resulting in a phosphorothioate (PS) bond. This substitution enhances the resistance of RNA to nuclease degradation and improves pharmacokinetic properties by increasing binding affinity to serum proteins such as albumin.
However, the introduction of PS bonds introduces chirality at the phosphorus center, leading to the formation of diastereoisomers with distinct biological activities and properties. Traditionally, synthetic oligonucleotides with PS modifications are produced as mixtures of these diastereoisomers, which can complicate their characterization and reduce therapeutic efficacy.
Advancements in synthetic chemistry have enabled the production of stereopure PS oligonucleotides, where the configuration of each phosphorus center is precisely controlled. Utilizing chiral phosphoramidite building blocks and optimized synthesis protocols, researchers have successfully synthesized fully stereodefined PS-modified RNA molecules.
For instance, in efforts to correct splicing defects associated with erythropoietic protoporphyria (EPP), fully stereopure 2'-O-(2-methoxyethyl)-phosphorothioate (PS-MOE) splice-switching oligonucleotides (SSOs) were developed. These SSOs, composed entirely of Rp-configured PS linkages, demonstrated superior hybridization affinity and enhanced splice-correction efficacy compared to their Sp counterparts in cellular assays targeting the ferrochelatase (FECH) gene.
The ability to synthesize stereopure PS oligonucleotides holds significant promise for improving the specificity and potency of RNA-based therapeutics by minimizing off-target effects and optimizing biological activity.
Terminal Phosphate Modifications
Both the 3'- and 5'-termini of RNA can be functionalized with various groups for analytical and therapeutic purposes. For instance, 3'-phosphorothioate monoesters have been used in template-dependent chemical ligations, which are facilitated by appropriately modified controlled-pore glass (CPG) solid supports. As RNA-based therapeutics advance in the clinic, reliable quantification techniques become essential. While methods like stem-loop RT-qPCR are standard for quantifying small RNAs, they are not suitable for all antisense therapeutics. One alternative is chemical ligation qPCR (CL-qPCR), which circumvents the need for enzymatic conversion of RNA to DNA.
In addition to 3'-phosphates, 2'3'-cyclic phosphate-terminated RNAs (RNA>p) play crucial roles in various biological processes, such as tRNA splicing and snRNA 3'-uridylation. Recent advances have enabled the synthesis of cyclic oligoadenylates, a novel class of second messengers in type III CRISPR-Cas systems. These molecules were identified through a combination of synthetic chemistry and enzymatic assays, highlighting the potential of RNA modifications in understanding cellular signaling pathways.
Chemical 5'-phosphorylation of synthetic RNAs is another area of interest, particularly for the preparation of long RNAs with site-specific modifications. A novel approach involves the synthesis of a dinitrobenzhydryl (DNB) containing phosphoramidite, which is compatible with RNA synthesis and purification. This method has been used to synthesize bis-labeled RNAs, which are valuable tools for structural studies on complex RNAs. Additionally, this chemistry has been applied in dynamic hyperpolarization solid-state NMR studies, where it has provided insights into surface reactions and RNA-protein interactions.
Ribose Modifications
The ribose sugar in RNA can be modified to introduce non-natural functionalities, creating tools for RNA chemical biology. Modified RNAs can be synthesized using various methods, including in vitro transcription and solid-phase synthesis. Site-specific modifications can be introduced either pre- or post-synthesis. One widely used method is the copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC), also known as "click chemistry." This approach is advantageous due to its biorthogonality, fast reaction rates, and compatibility with aqueous environments.
However, the introduction of artificial labels often impedes the biological activity of RNA. To address this, systematic investigations have been conducted to identify optimal labeling positions in pre-miRNAs. For example, labels in the terminal loop region of pre-miRNA hairpins or the 3p arm of 5p miRNAs are generally well-tolerated, while labels in the central region of the 5p strand or near the DICER processing site are not.
The growing interest in structured long non-coding RNAs has also driven the development of multi-labeled long RNAs. These RNAs are essential for RNA-capturing probes used in targetome identification of specific non-coding RNAs. The miRNA Crosslinking and Immunoprecipitation (miR-CLIP) technique, for instance, relies on dual-labeled miRNA precursors to elucidate the targetomes of disease-relevant miRNAs.
Modifications of the Nucleobase
Nucleobase modifications in RNA play a critical role in translational regulation and have found applications in nucleic acid-based drugs and biotechnology. One approach to obtaining base-modified oligonucleotides is the "convertible nucleoside" strategy. This method involves incorporating a nucleoside derivative containing a leaving group on the base into the RNA via solid-phase synthesis, followed by site-specific functionalization through substitution of the leaving group.
Since the introduction of this approach, various leaving groups specific to different positions on pyrimidines and purines have been reported. These modifications are useful for creating RNA-based tools with tailored functionalities. For example, modified nucleosides can be used to create RNA probes for detecting specific RNA-protein interactions, studying RNA structure, or modulating RNA function in cells.
Convertible nucleoside chemistry has also been applied to the synthesis of antisense oligonucleotides, where site-specific modifications can enhance therapeutic efficacy. By introducing modifications at strategic positions, researchers can improve the stability, binding affinity, and nuclease resistance of these oligonucleotides, making them more effective in clinical applications.
Conclusion
Advancements in RNA modification chemistry have significantly expanded the toolkit available for studying and manipulating RNA molecules. Modifications of the phosphodiester backbone, terminal phosphates, ribose sugar, and nucleobases have enabled the design of RNA constructs with enhanced stability, specificity, and functionality.
These chemical innovations have propelled the development of effective RNA-based therapeutics, such as antisense oligonucleotides and siRNAs, which hold promise for treating a wide range of diseases by modulating gene expression pathways. Additionally, sophisticated labeling and modification strategies have provided powerful tools for dissecting RNA biology, allowing detailed exploration of RNA structures, dynamics, and interactions in complex biological systems.
Continued research in RNA modification chemistry will undoubtedly yield further advancements, enabling more precise and effective therapeutic interventions and deeper insights into the fundamental roles of RNA in life processes. As our understanding and control over RNA chemistry improve, so too will our ability to harness RNA for innovative applications in medicine, biotechnology, and beyond.
References
- Röthlisberger P., et al. RNA chemistry for RNA biology. Chimia. 2019, 73 (5): 368-368.
- Imig J., et al. miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction. Nature Chemical Biology. 2015, 11 (2): 107-14.
- Flamme M., et al. Chemical methods for the modification of RNA. Methods. 2019, 161:64-82.
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