N3-Methyluridine and 2′-O-Alkyl/2′-Fluoro-N3-methyluridine functionalized nucleic acids improve nuclease resistance while maintaining duplex geometry

2′-O-Alkyl-N3-Methyluridine Functionalized Passenger Strand Improves RNAi Activity by Modulating the Thermal Stability

“…The observed lower T m value trend suggests that the N 3 -methyl group is removing the H-bond donor, potentially disrupting the Watson–Crick hydrogen bonding interactions and intrastrand stacking ( see molecular modeling section ). This observation correlates with the previously reported m 3 U or m 3 C modifications in RNA 14-mer or 12-mer sequences, leading to the weakening of hydrogen bonds, as evidenced by an increase in both average bond distance and fluctuations. , …”

“…This observation correlates with the previously reported m 3 U or m 3 C modifications in RNA 14-mer or 12-mer sequences, leading to the weakening of hydrogen bonds, as evidenced by an increase in both average bond distance and fluctuations. 14,23 Furthermore, the T m of siRNA duplexes was increased by 5.1 and 1.6 °C when 2′-OMe-m 3 U modifications were combined with multiple 2′-F-U modifications in the passenger strand (si-8 and si-9, respectively). Since the 2′-F modification is known to enhance binding affinity in RNA duplexes, these results indicate that incorporating 2′-alkoxy-m 3 U modifications, along with other 2′-modifications, may effectively counteract the thermal destabilization of siRNA duplexes.…”

“…26 Our previous report suggested that introducing a single 2′-alkoxym 3 U modification at either the 3′-or 5′-terminus of oligonucleotide, significantly enhances enzymatic stability in SVPD or PDE-II, respectively. 23 This success prompted us to investigate the influence of these modifications on the nuclease-mediated degradation of the modified siRNA strands. To evaluate the impact of these modifications against exonuclease-mediated degradation, we subjected the 3′penultimate modified passenger strands to snake venom phosphodiesterase (SVPD), a 3′-specific exonuclease.…”

“…The 2′-OMe-m 3 U, 2′-OEt-m 3 U, 2′-OMe-U, and 2′-F-U phosphoramidites were synthesized employing our previously established protocols. 23 The siRNAs targeting Renilla luciferase and endogenous Bcl-2 genes were synthesized by incorporating these modifications at overhangs, seed regions, and cleavage sites using an automated DNA/RNA synthesizer following the conventional phosphoramidite protocols. The siRNA sequences are highlighted in Table S1.…”

“…Our laboratory has reported the synthesis of a series of 2′-alkoxy/fluoro- N 3 -methyluridine nucleosides including 2′-F-m 3 U, 2′-OMe-m 3 U, 2′-OEt-m 3 U, 2′-OPr-m 3 U, and 2′-OMOE-m 3 U. , The incorporation of these modified monomers at the central position in 12-mer and 14-mer oligonucleotides exhibited a substantial reduction in melting temperature (around 8–12 °C per modification). Moreover, nuclease resistance studies showed that 2′- O -alkyl-m 3 U modifications improved the half-life of oligonucleotides against 3′- and 5′- exonucleases as compared to 2′-fluoro and 2′-OMe modified oligonucleotides.…”