What are antisense oligonucleotides (ASOs)?

Antisense oligonucleotides (ASOs) are short nucleic acids designed to bind to specific RNA sequences, thereby inhibiting, reducing, restoring, or modifying the expression of target RNA. Over the past two decades, this method of gene silencing at the posttranscriptional stage has become increasingly popular as a therapeutic technology, receiving FDA approval for treating conditions such as muscular and spinal atrophy.

Why is mesyl phosphoramidate being used?

The first generation of modified ASOs aimed to protect the natural phosphodiester bond of the backbone against nuclease digestion. This was achieved by using phosphorothioate oligo-deoxynucleotides (PS-ODNs), where the non-bridging oxygen atom is substituted with a sulfur atom. While this modification increased nuclease resistance and facilitated RNase H recruitment to cleave the RNA strand, PS-modified oligos presented challenges such as insufficient biological activity, off-target toxic effects, and low target engagement.

Recent studies have highlighted a new type of modification, mesyl-phosphoramidate antisense oligonucleotides (ASOs), which was used to target the pro-oncogenic miR-21 in melanoma B16 cells and in a xenograft mouse tumor model [1,2]. The results suggested the modification increases ASOs' stability in biological environments, facilitates cellular uptake, and enhances intracellular trafficking. With improved pharmacokinetic properties and target engagement, mesyl modification holds promise as a replacement for phosphorothioate (PS) in future antisense oligonucleotide applications, pending further studies.

What is mesyl and how is it synthesized?

Mesyl phosphoramidate oligonucleotides are DNA analogs that replace the natural phosphodiester group with a mesyl (methanesulfonyl) group (known by µ-modification or MsPA).

Image 1: Chemical structure of phosphorothioate (PS) and mesyl phosphoramidate that can replace phosphodiester bond (PO)

These oligonucleotides can be synthesized using the traditional solid-phase synthesis method with controlled pore glass or polymer-based support. Synthesis of µ-modification oligos and PS-ODNs follows similar stages but differs in the oxidation step, where an aqueous iodine oxidizer is replaced with 0.5-1 M methanesulfonyl azide in acetonitrile (or other compatible solvents such as ACN or toluene). The Staudinger reaction occurs between the support-bound phosphite triester and mesyl azide within 15-30 minutes, emitting N2. After synthesis, oligonucleotides are cleaved from the support and deprotected using ammonia treatment at 55°C. Purification is carried out using reverse-phase HPLC or conventional gel electrophoresis (PAGE), with purity analysis confirmed by ESI LC-MS or MALDI-TOF MS.

Similarly, close analogs of mesyl phosphoramidate can be synthesized using the same strategy. Caution should be exercised due to the generation of one molecule of nitrogen for every reaction. Avoid recirculating the reaction in a closed-loop system; instead, opt for flow-through mode.

What are the common impurities associated with this modification?

Due to the slow reaction kinetics of the Staudinger reaction, incomplete reactions or hydrolysis can lead to an impurity of -77.1 Da, like PS to PO conversion.

References:

1. Miroshnichenko, S. K., Patutina, O. A., Burakova, E. A., Chelobanov, B. P., Fokina, A. A., Vlassov, V. V., Altman, S., Zenkova, M. A., & Stetsenko, D. A. (2019). Mesyl phosphoramidate antisense oligonucleotides as an alternative to phosphorothioates with improved biochemical and biological properties. Proceedings of the National Academy of Sciences, 116(4), 1229–1234. https://doi.org/10.1073/pnas.1813376116
2. Patutina, O. A., Gaponova Miroshnichenko, S. K., Sen'kova, A. V., Savin, I. A., Gladkikh, D. V., Burakova, E. A., Fokina, A. A., Maslov, M. A., Shmendel', E. V., Wood, M. J. A., Vlassov, V. V., Altman, S., Stetsenko, D. A., & Zenkova, M. A. (2020). Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency. Proceedings of the National Academy of Sciences of the United States of America, 117(51), 32370–32379. https://doi.org/10.1073/pnas.2016158117

Why is TNA being used?

The use of oligonucleotides in therapeutics and diagnostics is rapidly expanding, particularly in the realm of antisense therapy. In previous discussions, we explored how modifying the backbone of antisense oligonucleotides (ASOs) with Mesyl can enhance stability and therapeutic efficacy. In this blog, we shift our focus to a promising class of artificial genetic polymers known as Xeno-nucleic acids (XNAs), with specific emphasis on α-L-Threose nucleic acid (TNA). TNA has attracted significant interest due to its structural simplicity and unique ability to form stable duplex structures with complementary DNA and RNA strands.

Various studies have investigated the biological properties and potential of TNA as antisense agents, including inhibition of GFP gene expression, detection of target miRNAs, and real-time monitoring of miRNA expression levels in cell lines [3,4,5]. Results indicate that TNA exhibits excellent resistance to acid degradation, high stability at room temperature storage, low cytotoxicity, and significant uptake in various cell lines without carriers. While further studies are necessary to fully validate the benefits of TNA, it remains an attractive candidate for biomedical and therapeutic applications.

What is TNA and how is it synthesized?

TNA, or threose nucleic acid, represents a synthetic analog of DNA and RNA, featuring threofuranosyl nucleotides and a backbone comprised of threose sugar units, threofuranose, instead of the ribose or deoxyribose typically found in DNA and RNA. Unlike the conventional 3'- to 5'-linkage in DNA and RNA, the phosphate group in TNA connects at the 2' to 3'-carbon. To synthesize TNA oligonucleotides, TNA phosphoramidite monomers of adenine (A), guanine (G), cytosine (C), and thymine (T)/uracil (U)) are utilized. The synthesis process follows Eschenmoser’s procedure, involving glycosylation, debenzoylation, tritylation, and phosphitylation steps. After stable pre-drying, the monomers are dissolved in acetonitrile and utilized in the solid-phase synthesis method using controlled pore glass. To ensure high purity and yield, the frequency of de-blocking and coupling reactions is increased, along with the reaction time in each synthesis cycle. Post-synthesis purification is conducted using C18 reverse-phase HPLC or conventional polyacrylamide gel electrophoresis (PAGE), with purity and molecular weight analysis confirmed by MALDI-TOF MS.

Image 1: Chemical structure of DNA, RNA and TNA oligonucleotide, where α-L-threose nucleic acid (TNA) has one less atom in its backbone unit and has 2´, 3´ phosphodiester linkage

Does Synoligo synthesize TNA?

Yes, we have over 200+ modifications you can choose from for your project including TNA and TNA-U (Bz).

Feel free to contact us if you have any questions about starting a project with us.

References:

1. Zhang, S., & Chaput, J. C. (2012). Synthesis of Threose Nucleic Acid (TNA) Phosphoramidite Monomers and Oligonucleotide Polymers. Current Protocols in Nucleic Acid Chemistry, 4.51.1–4.51.26. doi:10.1002/0471142700.nc0451s50
2. Bege, M.; Borbás, A. The Medicinal Chemistry of Artificial Nucleic Acids and Therapeutic Oligonucleotides. Pharmaceuticals 2022, 15, 909. https://doi.org/10.3390/ph15080909
3. Liu, L. S., Leung, H. M., Tam, D. Y., Lo, T. W., Wong, S. W., & Lo, P. K. (2018). α-l-Threose Nucleic Acids as Biocompatible Antisense Oligonucleotides for Suppressing Gene Expression in Living Cells. ACS Applied Materials & Interfaces, 10(11), 9736–9743. doi:10.1021/acsami.8b01180
4. Wang, F., Liu, L. S., Li, P., Lau, C. H., Leung, H. M., Chin, Y. R., Tin, C., & Lo, P. K. (2022). Cellular uptake, tissue penetration, biodistribution, and biosafety of threose nucleic acids: Assessing in vitro and in vivo delivery. Materials today. Bio, 15, 100299. https://doi.org/10.1016/j.mtbio.2022.100299
5. Wang, F., Liu, L. S., Li, P., Leung, H. M., Tam, D. Y., & Lo, P. K. (2022). Biologically stable threose nucleic acid-based probes for real-time microrna detection and imaging in living cells. Molecular Therapy - Nucleic Acids, 27, 787–796. https://doi.org/10.1016/j.omtn.2021.12.040