Role of a “Magic” Methyl: 2′-Deoxy-2′-α-F-2′-β-C-methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5′-Phosphate Mimics and Mitigation of Off-Target Effects

Although 2′-deoxy-2′-α-F-2′-β-C-methyl (2′-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2′-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3′-endo preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2′-F/Me modification imparted more resistance to degradation than the corresponding 2′-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2′-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary N-acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2′-F/Me modification was combined with 5′-vinyl phosphonate, both E and Z isomers had silencing activity comparable to the parent. In combination with other 2′-modifications such as 2′-O-methyl, the Z isomer is detrimental to silencing activity. Presumably, the equivalence of 5′-vinyl phosphonate isomers in the context of 2′-F/Me is driven by the steric and conformational features of the C-methyl-containing sugar ring. These data indicate that 2′-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.