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Coenzyme A overview

Modified Nucleosides: Functions, Synthesis, and Medical Applications

Overview of Modified Nucleosides

Modified nucleosides are chemically altered versions of the standard building blocks of RNA and DNA [1,2]. These modifications occur naturally through post-transcriptional processes, in which chemical changes are made to RNA after it has been synthesized within a cell [1,2]. Scientists have identified over 100 different types of RNA modifications, ranging from simple chemical substitutions to more complex structures [1,2]. These modifications play critical roles in RNA structure and function, contributing to the stability of RNA molecules and supporting accurate and efficient protein synthesis [2]. In addition to their biological roles, nucleoside chemistry forms the basis for the chemical synthesis of DNA and RNA used in research and medical applications [3].

Biological Function of Modified Nucleosides in RNA

In living cells, these modifications are vital for the structure, stability, and function of RNA molecules [1,2]. In tRNA and ribosomal RNA (rRNA), they contribute significantly to the accuracy and efficiency of protein translation [2,4]. Modifications in the anticodon region directly influence decoding accuracy and reading frame maintenance; without them, cells struggle to translate genetic information effectively [2,5]. Furthermore, these chemical changes help tRNAs fold into their critical three-dimensional L-shape and produce unique recognition determinants for other macromolecules [2,6].

Synthesis & Applications of Modified Nucleosides & Nucleotides 

Natural nucleotides are the fundamental building blocks of nucleic acids and are essential for all living organisms. Research into biological systems has revealed a wide range of naturally occurring nucleotide modifications and their important roles in regulating bioprocesses, as well as their links to human diseases [7]. Nucleoside analogues and their phosphorylated prodrugs have been widely studied for their antiviral [8] and antitumor [9] properties [10,11]. Emerging fields exploring the therapeutic potential of modified nucleic acids include RNA vaccines, antisense oligonucleotides (ASOs), and small interfering RNA (siRNA). 

 

Advances in diagnostics, bioprocess monitoring, and nucleic acid therapeutics have increased the demand for versatile methods of nucleic acid modification and labeling. Modifications can be introduced at any key component of the nucleotide structure, including the ribose ring, nucleobase, or phosphate group.

 

Functionalized or labeled nucleic acids are commonly synthesized via two main strategies. The first is solid-phase synthesis, which enables routine preparation of unmodified or modified oligonucleotides (ONs) of up to ~100–200 nucleotides using automated chemical synthesis of oligonucleotides [8,12-14]. Phosphoramidite chemistry typically employs acid-labile DMT (4,4′-dimethoxytrityl) protecting groups for the 5′-hydroxyl and base-labile groups such as benzoyl (bz) or isobutyryl (ib) for nucleobases [15,16]. 

 

The second approach to preparing modified nucleic acids is enzymatic incorporation of modified 5′-triphosphates using polymerases. Copper-catalyzed or strain-promoted azide-alkyne cycloaddition (CuAAC, SPAAC) provides highly flexible reaction conditions, including aqueous, organic, or mixed solvent systems. Orthogonal azide and alkyne groups are nearly inert under physiological conditions due to their weak acid-base properties.This approach is widely used in bioconjugation and won the 2022 Nobel Prize in Chemistry for the development of “click chemistry” and bioorthogonal chemistry [17]. 

 

Modified nucleotides are essential tools in cutting-edge molecular biology applications, including fluorescence in situ hybridization (FISH) [18], systematic evolution of ligands by exponential enrichment (SELEX) [12], next-generation sequencing (NGS) [19], and many other techniques as well as in the development of pharmaceuticals [10] and RNA vaccines [20]. 

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Product Spotlights

  • 5'-Azido-5'-deoxythymidine: This modified nucleoside derived from thymidine is distinguished by an azide group "tag" at the 5' end [21,22]. In biotechnology, it is a critical tool for "click chemistry", acting as a reactive site to join DNA strands together [23]. 

  • 5'-Deoxy-5'-iodothymidine: This modified nucleoside serves as a critical synthetic intermediate in oligonucleotide synthesis [23,24]. These iodo groups are essential for subsequent conversion into azides, allowing for the direct incorporation of functional sites into oligonucleotides for click ligation [23]. 

  • DMT-Protected Compounds: These derivatives act as primary "bodyguards" during DNA synthesis [25]. DMT is the standard acid-labile group used to block the 5'-hydroxyl position, and its quantitative removal in each cycle is vital for high-quality DNA [25,26]. Notably, 5'-O-DMT-thymidine is the most stable of the four standard building blocks in acetonitrile solutions, while DMT-dG is the most susceptible to degradation [26,27]. 

  • 5',3'-TIPS 5-methyluridine: This modified nucleoside utilizes a TIPS (or TIPDS) "selective shield" to simultaneously protect the 3' and 5' positions of the sugar [28]. By locking these positions, the 2'-hydroxyl is left available for specific chemical modifications, such as the radical deoxygenation required to convert RNA components into DNA-like building blocks [28,29]. 

  • 5'-Iodo Protected Deoxynucleosides (G and C): 5'-Iodo-N2-isobutyryl-2′-deoxyguanosine and 5'-Iodo-N4-benzoyl-2'-deoxycytidine are "click-ready" versions of guanine and cytosine [23,26]. These modified nucleosides use isobutyryl and benzoyl groups to protect the nucleobase during synthesis [26, 30]. Like their thymidine counterparts, the 5'-iodo tag on these molecules allows scientists to join different strands of DNA with precision using copper-catalyzed click chemistry [23]. 

Modified Nucleosides in Disease and Drug Development

Modified nucleoside analogs are cornerstones of modern medicine. Approved drugs include Zidovudine (AZT) for HIV, Cladribine for leukemia, and Idoxuridine for viral infections [31-33]. Mutations in modification enzymes are linked to severe pathologies:

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  • Metabolic Disorders: Mutations in the Cdkal1 protein result in decreased insulin secretion and Type 2 diabetes [2,34]. 

  • Mitochondrial Syndromes: The absence of specific mitochondrial modifications is implicated in MELAS and MERRF syndromes [34,35]. 

  • Neurological Impact: Mutations in RNA-editing enzymes known as ADARs are linked to cancer and neurological disorders [2,36]. 

  • Antibacterial Resistance: Changes in the methylation status of 16S rRNA, a key structural component of the bacterial ribosome involved in protein synthesis, allow pathogenic bacteria to develop resistance to aminoglycoside antibiotics [2,37].

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Modified Nucleosides as Cancer Biomarkers

Modified nucleosides serve as promising urinary tumor biomarkers because neoplastic (cancerous) tissues exhibit a much more rapid RNA turnover rate and contain hyperactive methyltransferases than healthy tissues [38,39]. Significant elevations in nucleosides such as cytidine, pseudouridine, 1-methyladenosine, and PCNR provide a reliable biochemical indicator for early cancer detection [38,40]. 

References

  1. Russell, S. P.; Limbach, P. A. Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS. J. Chromatogr. B 2013, 923–924, 74–82. 

  2. Agris, P. F.; Väre, V. Y. P. et al. Chemical and Conformational Diversity of Modified Nucleosides Affects tRNA Structure and Function. Biomolecules 2017, 7 (29). 

  3. Caruthers, M. H. A brief review of DNA and RNA chemical synthesis. Biochem. Soc. Trans. 2011, 39, 575–580. 

  4. Björk, G. R. et al. A modification at the wobble position of tRNA 1 stops cleavage by angiogenin and is required for viability of Saccharomyces cerevisiae. FEBS Lett. 1999, 452, 47–51. 

  5. Ashraf, S. S. et al. Single modification at the wobble position of human tRNALys3 allows for accurate decoding of the HIV-1 genome. RNA 1999, 5, 503–511. 

  6. Vendeix, F. A. et al. Human tRNALys3UUU is pre-structured by natural modifications for cognate and wobble codon binding through keto-enol tautomerism. Biochemistry 2008, 47, 6117–6129. 

  7. P. J. McCown, A. Ruszkowska, C. N. Kunkler, K. Breger, J. P. Hulewicz, M. C. Wang, N. A. Springer and J. A. Brown, "Naturally occurring modified ribonucleosides," WIREs RNA, p. e1595, 2020. 

  8. E. D. Clercq, "The history of antiretrovirals: key discoveries over the past 25 years," Rev. Med. Virol., vol. 19, p. 287–299, 2009. 

  9. M. Guinan, C. Benckendorff, M. Smith and G. J. Miller, "Recent Advances in the Chemical Synthesis and Evaluation of Anticancer Nucleoside Analogues," Molecules, vol. 25, no. 9, p. 1–25, 2020. 

  10. Y. Zhang, Y. Gao, X. Wena and H. Ma, "Current prodrug strategies for improving oral absorption of nucleoside analogues," Asian J. Pharm. Sci., vol. 9, no. 2, p. 65−74, 2014. 

  11. S. Mahmoud, S. Hasabelnaby, S. F. Hammad and T. M. Sakr, "Antiviral Nucleoside and Nucleotide Analogs: A Review," J. Adv. Pharm. Res., vol. 2, no. 2, p. 73–88, 2018. 

  12. A. D. Keefe and S. T. Cload, "SELEX with modified nucleotides," Curr Opin Chem Biol, vol. 12, pp. 448-456, 2008. 

  13. D. E. Chapman and G. Powis, "Disposition and metabolism in mice of the potential antitumor and anti-human immunodeficiency virus-1 agent, 2-chloro-2',3'-dideoxyadenosine," Cancer Chemother. Pharmacol., vol. 27, pp. 285-289, 1991. 

  14. H. Lonnberg, "Solid-Phase Synthesis of Oligonucleotide Conjugates Useful for Delivery and Targeting of Potential Nucleic Acid Therapeutics," Bioconjugate Chem., vol. 20, no. 6, pp. 1065-1094, 2009. 

  15. U. Pradere, E. C. Garnier-Amblard, S. J. Coats and F. Amblard, "Synthesis of Nucleoside Phosphate and Phosphonate Prodrugs," Chem. Rev., vol. 114, pp. 9154-9218, 2014. 

  16. A. D. Barone, C. Chen, G. H. McGall, K. Rafii, P. R. Buzby and J. J. Dimeo, "Novel Nucleoside Triphosphate Analogs For The Enzymatic Labeling Of Nucleic Acids," Nucleosides, Nucleotides and Nucleic Acids, vol. 20, pp. 1141-1145, 2001. 

  17. L. Liang and D. Astruc, "The copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) “click” reaction and its applications. An overview," Coord. Chem. Rev., vol. 255, pp. 2933-2945, 2011. 

  18. S. Gad, A. Aurias, N. Puget, A. Mairal, C. Schurra, M. Montagna, S. Pages, V. Caux, S. Mazoyer, A. Bensimon and D. Stoppa-Lyonnet, "Color bar coding the BRCA1 gene on combed DNA: A useful strategy for detecting large gene rearrangements," GenesChromosom. Cancer, vol. 31, pp. 75-84, 2021. 

  19. M. L. Metzker, "Sequencing technologies — the next generation," Nat. Rev., vol. 11, p. 31–46 , 2010 

  20. C. Pollard, S. D. Koker, X. Saelens, G. Vanham and J. Grooten, "Challenges and advances towards the rational design of mRNA vaccines," Trends Mol. Med., vol. 19, no. 12, p. 705–713, 2013. 

  21. Hiebl, J. et al. Synthesis and Antiretrovirus Properties of 5’-Isocyano-5’-deoxythymidine and related analogues. J. Med. Chem. 1991, 34, 1426–1430. 

  22. Hiebl, J.; Zbiral, E. Glycosyl azides as starting materials for the preparation of nucleoside analogues. Liebigs Ann. Chem. 1988, 765. 

  23. El-Sagheer, A. H. et al. Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli. Proc. Natl. Acad. Sci. USA 2011, 108, 11338–11343. 

  24. Miller, G. P.; Kool, E. T. Versatile 5′-functionalization of oligonucleotides on solid support: Amines, azides, thiols, and thioethers via phosphorus chemistry. J. Org. Chem. 2004, 69, 2404–2410. 

  25. Septak, M. Kinetics of acid-catalyzed detritylation of solid-supported oligonucleotides. Nucleic Acids Res. 1996, 24, 3053–3058. 

  26. Krotz, A. H. et al. Solution Stability and Degradation Pathway of Deoxyribonucleoside Phosphoramidites in Acetonitrile. Nucleosides, Nucleotides Nucleic Acids 2004, 23 (5), 767–775. 

  27. Capaldi, D. C. et al. Purification of oligonucleotides using a novel approach to enhance purity. Org. Process Res. Dev. 2003, 7, 832–838. 

  28. Markiewicz, W. T. Tetraisopropyldisiloxane-1,3-diyl, a hindered bifunctional protecting group for simultaneous protection of 3' and 5' hydroxyl groups of ribonucleosides. J. Chem. Res., Synop. 1979, 24. 

  29. Robins, M. J.; Wilson, J. S.; Hansske, F. Nucleic Acid Related Compounds. 42. A General Procedure for the Efficient Deoxygenation of Secondary Alcohols. J. Am. Chem. Soc. 1983, 105, 4059–4065. 

  30. Stengele, K. P. et al. Diarylsulfide photolabile protecting groups. U.S. Patent 11,001,602 B2, May 11, 2021. 

  31.  Shen, B.; Jamison, T. F. Continuous Flow Photochemistry for the Rapid and Selective Synthesis of 2’-Deoxy and 2’,3’-Dideoxynucleosides. Aust. J. Chem. 2013, 66*, 157–164. 

  32. Ichikawa, E.; Kato, K. 2'-Deoxynucleoside synthesis: An updated review. Curr. Med. Chem. 2001, 8, 385–423. 

  33. Xia, R.; Chen, L. S. Efficient Synthesis of Cladribine via the Metal-Free Deoxygenation. Nucleosides, Nucleotides Nucleic Acids 2015, 34, 1–7. 

  34. Agris, P. F. et al. tRNA modifications and Type 2 diabetes. RNA 2015, 21, 552–554. 

  35. Suzuki, T.; Suzuki, T. Taurine modification of mitochondrial tRNAs and human diseases. Nucleic Acids Res. 2014, 42, 7346–7357. 

  36. Zinshteyn, B.; Nishikura, K. ADAR RNA editing. Wiley Interdiscip. Rev. Syst. Biol. Med. 2009, 1, 202–209. 

  37. Shakil, S. et al. 16S rRNA methylation and aminoglycoside resistance in pathogenic bacteria. J. Biomed. Sci. 2008, 15, 5–18. 

  38. Seidel, A. et al. Modified nucleosides: an accurate tumour marker for clinical diagnosis of cancer, early detection and therapy control. Br. J. Cancer 2006, 94, 1726–1733. 

  39. Schöch, G. et al. In Chromatography and Modification of Nucleosides Part C; Elsevier: Amsterdam, 1990; pp C389–C442. 

  40. Zheng, Y. F. et al. Urinary modified nucleosides as tumor markers in breast cancer. Clin. Biochem. 2005, 38, 24–30. 

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