Conjugation and Evaluation of Small Hydrophobic Molecules to Triazole-Linked siRNAs

Abstract

Short interfering RNAs (siRNAs) have tremendous potential as a new class of next-generation therapeutics; however, their progress is lagging due to issues related to stability, biodistribution, and cell-membrane permeability. To overcome these issues, there is widespread interest in chemically modifying siRNAs. In this study, siRNAs that contain a triazole-backbone unit with pyrimidine-modified hydrophobic substituents were synthesized and examined for their gene-silencing activity. In our study, we generated a library of siRNAs that target both a plasmid reporter system and an endogenous gene target, bcl-2. Our results indicate that these unique modifications are well tolerated within the RNA interference pathway. In addition, a cholesterol-modified triazole-linked siRNA targeting the exogenous target firefly luciferase was capable of gene-silencing at levels greater than 80% in the absence of a carrier complex.

siRNAzos: A New Class of Azobenzene‐Containing siRNAs that Can Photochemically Regulate Gene Expression

“…This is consistent with other studies that place thermally destabilizing units within siRNAs , − Finally, the siRNAzos all retained classic A‐type conformation, which is necessary for the RNAi pathway, as demonstrated by circular dichroism (Figure S‐1 in the Supporting Information).…”

Effective carrier-free gene-silencing activity of cholesterol-modified siRNAs

“…2, the cholesterol-modied triazole-linked siRNAs (X1, X2, and X5) exhibit potent gene silencing, with 70-80% reduction in rey luciferase activity in the 500 to 3000 nM concentration range. As previously reported, placing a chemical modication within the central region of the sense strand may impact thermal destabilization, [22][23][24] however, this does not seem to alter genesilencing efficacy. In fact, the IC 50 s for these thermallydestabilized centrally-modied siRNAs X1 and X2 were 243.6 nM and 307.1 nM respectively.…”

Effective gene-silencing of siRNAs that contain functionalized spacer linkages within the central region

“…Prior work from our laboratory involving an siRNA bearing a cholesterol-derivatized triazole moiety also showed signicant thermal destabilization, despite a favorable gene silencing biological response. 17 We next turned our attention to the derivatized spacer molecules that replace two consecutive nucleoside derivatives. With respect to exible aliphatic spacers spanning positions 9 and 10 of the sense strand, we observed dose dependent genesilencing with siRNAs 28, 29, and 30, which contain two acetyl groups, two propargyl groups, and two allyl groups, respectively ( Fig.…”