siRNA Modified with 2′‐Deoxy‐2′‐C‐methylpyrimidine Nucleosides

Abstract

(2'S)-2'-Deoxy-2'-C-methyluridine and (2'R)-2'-deoxy-2'-C-methyluridine were incorporated in the 3'-overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2'S)-2'-deoxy-2'-C-methylcytidine was incorporated in the 6-position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3'-overhang region by (2'S)- or (2'R)-2'-deoxy-2'-C-methyluridine, reducing IC to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.

Chemical strategies for strand selection in short-interfering RNAs

“…4 ) were evaluated for their influence on strand specific activity. 90 These modifications were tested at positions AS:5, AS:6 AS:20 and SS:20. Modification of position AS:20 with either R or S configuration was found to improve the IC 50 .…”

Chemical Modifications in Nucleic Acids for Therapeutic and Diagnostic Applications

“…[76] Another conformationally restricted nucleotides investigated by our group were 2'-deoxy-2'-C-methylpyrimidine nucleosides (Figure 3). [77] Specifically, (2'S)-2'-Deoxy-2'-Cmethyluridine, (2'R)-2'-deoxy-2'-C-methyluridine and (2'S)-2'-deoxy-2'-C-methylcytidine were incorporated in the 3'overhang regions of the sense and antisense strands and in the seed region of siRNA duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3'overhang region by (2'S)-or (2'R)-2'-deoxy-2'-C-methyluridine, increasing the inhibitory potency twice of the value observed for the unmodified siRNA control.…”

“…Another parameter that was studied was the possibility of incorrect strand selection by RISC using specific plasmids that can measure the silencing activity of the guide or the passenger strand. It was found that the presence of 2’‐deoxy‐2’‐ C ‐methylpyrimidine nucleosides can increase the selectivity of the modified siRNA, especially when these modifications were located in positions 5 and 6 of the seed region [77] …”

“…It was found that the presence of 2'-deoxy-2'-C-methylpyrimidine nucleosides can increase the selectivity of the modified siRNA, especially when these modifications were located in positions 5 and 6 of the seed region. [77] Although conformationally restricted nucleotides could be used in RNA interference as they increase thermal stability and nuclease resistance, researchers also found that the presence of flexible nucleosides such as acyclic nucleosides [78,79] and especially Unlocked Nucleic Acids [80,81] may be beneficial for RNA interference. In the previous section, it was already mentioned that the presence of acyclic threoninol nucleic acid (aTNA) units at the 3'-end of the guide strand was beneficial because the presence of the acyclic nucleoside at the 3'-end optimized the interactions of the guide strand with the PAZ domain of RISC.…”