A comprehensive analysis of the use of nucleoside analogues in RNA therapeutics

mRNA therapeutics represent a promising frontier in the development of vaccines, anti-cancer treatments, and gene replacement therapies. The mRNA platform facilitates the rapid design of effective and safe candidates. However, native mRNA molecules are inherently characterized by limited stability and high reactogenicity, which may compromise their in vivo efficacy. The incorporation of modified nucleoside analogues can address these challenges by extending the mRNA half-life and subsequently enhancing protein expression. Despite the discovery and widespread adoption of pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ) in mRNA therapeutics, these modifications have not fully addressed all inherent limitations of mRNA-based platforms. Consequently, identifying novel nucleoside analogues, their combinations, and alternative therapeutic strategies remains a critical area of research. This review provides a comprehensive overview of how commonly used nucleoside analogues influence mRNA stability, translation, and immunogenicity, while also discussing their natural distribution patterns in native mRNA. Furthermore, we examine experimental in vitro and in vivo data regarding the application of non-canonical nucleosides in cell and animal models and evaluate the future prospects of modified mRNA-based therapies. In conclusion, this review provides an extensive analysis of the properties of modified nucleosides employed in mRNA formulations and characterizes their impact on the overall efficacy of mRNA-based therapeutics in detail.