Locked 2′-Deoxy-2′,4′-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity

Abstract

2′-Deoxy-2′,4′-difluorouridine (2′,4′-diF-rU) was readily incorporated into DNA and RNA oligonucleotides via standard solid phase synthesis protocols. NMR and thermal denaturation (T m) data of duplexes was consistent with the 2′,4′-diF-rU nucleotides adopting a rigid North (RNA-like) sugar conformation, as previously observed for the nucleoside monomer. The impact of this modification on T m is neutral when incorporated within RNA:RNA duplexes, mildly destabilizing when located in the RNA strand of a DNA:RNA duplex, and highly destabilizing when inserted in the DNA strand of DNA:RNA and DNA:DNA duplexes. Molecular dynamics calculations suggest that the destabilization effect in DNA:DNA and DNA:RNA duplexes is the result of structural distortions created by A/B junctions within the helical structures. Quantum mechanics calculations suggest that the “neutral” effect imparted to A-form duplexes is caused by alterations in charge distribution that compensate the stabilizing effect expected for a pure North-puckered furanose sugar. 2′,4′-diF-RNA modified siRNAs were able to trigger RNA interference with excellent efficiency. Of note, incorporation of a few 2′,4′-diF-rU residues in the middle of the guide (antisense) strand afforded siRNAs that were more potent than the corresponding siRNAs containing LNA and 2′-F-ANA modifications, and as active as the 2′-F-RNA modified siRNAs.

Structural basis for the synergy of 4′- and 2′-modifications on siRNA nuclease resistance, thermal stability and RNAi activity

“…Earlier reports of 4′- C -modified nucleotides, including 4′- C -OMe-2′-deoxynucleotides ( 17 , 18 ) and 2′-F,4′- C -F pyrimidine nucleotides ( 19–21 ) and their incorporation into oligonucleotides support the notion of a preference for a Northern sugar pucker. The thermodynamic stability of RNA duplexes containing such nucleotides is similar to that of the native counterparts ( 21 ). Indeed, we found that incorporation of 2′-F,4′- Cα -OMe-U ( Ufo , Figure 1 ) caused only minor changes to the melting temperature ( T m ) of RNA duplexes, whereby the modified ribose adopted a North ( N ) C3′- endo conformation in the crystal structure of a modified octamer ( 16 ).…”

Therapeutic potential of chemically modified siRNA: Recent trends

“…It gave a mild destabilizing effect when incorporated in the RNA strand of DNA:RNA duplex and high destabilization when incorporated in the DNA strand of DNA:RNA and DNA:DNA duplex. Modification in the central region of guide strand with 2′,4′‐diF‐rU was more potent in silencing the gene expression …”

“…Modification in the central region of guide strand with 2′,4′-diF-rU was more potent in silencing the gene expression. [27] siRNAs modified with 2′-O-methyl guanosine and uridine residues profoundly decreased the immunostimulation and interferon response. The effect of modification by 2′-O-methyl-4′-thioRNA (10) on gene silencing was studied against HeLa-Luc gene.…”

“…AS Near cleavage site and seed region U 3 (+ to −) silencing activity relative to 2′-F-RNA and LNAs tm (−15.6 to +3.5) [27] 2′-OMe-4′-thioribonucleoside-mod ified siRNA (2′-OMe-4′-thioribo-siRNAs)…”

“…The cellular uptake ability was high when CPP was conjugated with siRNA and triggered by ultrasound. [44] Conjugation of siRNAs with aromatic compounds such as phenyl, hydroxyphenyl, naphthyl and pyrenyl derivatives (27) are protected from nuclease degradation. They were also proved to be highly stable thermodynamically.…”