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

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 Tm 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.