Modified DNA phosphoramidites and RNA phosphoramidites play a crucial role in research as they allow the introduction of specific chemical modifications into nucleic acids, enabling studies on structure-function relationships, gene expression regulation, and therapeutic applications such as gene editing and RNA interference (RNAi).
Recently, scientists (see Reference below) investigated the potential of RNA interference as a targeted gene silencing tool and its applications in gene technology and therapeutic development. RNAi is a natural defense mechanism that utilizes short interfering RNA (siRNA) to silence specific genes. These siRNAs form RNA-induced silencing complexes (RISC) that degrade targeted mRNA sequences, leading to gene silencing without causing non-specific RNA degradation. This specificity makes siRNA a powerful tool for studying biological functions and a potential therapeutic agent for various diseases.
The researchers synthesized three duplex RNAs, where modified nucleosides replaced unmodified nucleosides in the 3'-overhang region of the siRNA. The modified nucleosides were
designed based on the study of hepatocyte nuclear factor 4a (HNF4α), a nuclear hormone receptor involved in liver development and the expression of tissue-specific traits, to improve siRNA efficacy against the receptor. In vitro experiments showed that the siRNAs with modified nucleosides were more effective in suppressing HNF4α compared to unmodified siRNAs.
The synthesis of modified siRNAs involved the preparation of modified nucleosides and the subsequent assembly of the siRNA molecules (Figures 1 and 2). Phosphoramidite chemistry was employed to synthesize the modified nucleosides, which were then incorporated into the siRNA strands using automated DNA/RNA synthesizers. The resulting siRNAs were purified by high-performance liquid chromatography (HPLC) to ensure high purity.
Figure 1. Example of synthesis of a modified nucleoside phosphoramidite
Figure 2. One of the oligoribonucleotides synthesized for modified siRNA using 21 modified nucleosides, 19 of which are represented by
BroadPharm such as BP-28840,
BP-28839, BP-29973, BP-28837. The bottom two modified uridine bases in this strand's sequence are structurally different from the other three modified uridine bases.
Masud MM, Masuda T, Inoue Y, Kuwahara M, Sawai H, Ozaki H. Synthesis of modified siRNA bearing C-5 polyamine-substituted pyrimidine nucleoside in their 3'-overhang regions and its RNAi activity. Bioorg Med Chem Lett. 2011 Jan 15;21(2):715-7. doi: 10.1016/j.bmcl.2010.11.125. Epub 2010 Dec 5. PMID: 21190853.