A key approach for improving siRNA efficacy is chemical modifications. Through an in silico screening of modifications at the 5'-end nucleobase of the guide strand, an adenine-derived compound called 6-(3-(2-carboxyethyl)phenyl)-purine (6-mCEPh-purine) was identified to improve the RNAi activity in cultured human cells and in vivo mouse models. Nevertheless, it remains unclear how this chemical modification enhances the siRNA potency. Here, we used a series of biochemical approaches to quantitatively evaluate the effect of the 6-mCEPh-purine modification at each step in the assembly of the RNAi effector complex called RISC. We found that the modification improves the formation of mature RISC at least in two different ways, by fixing the loading orientation of siRNA duplexes and increasing the stability of mature RISC after passenger strand ejection. Our data will provide a molecular platform for further development of chemically modified siRNA drugs.
Adenine/pharmacology , Argonaute Proteins/genetics , RNA Interference/drug effects , RNA, Double-Stranded/genetics , RNA, Small Interfering/agonists , RNA-Induced Silencing Complex/agonists , Adenine/analogs & derivatives , Adenine/chemical synthesis , Argonaute Proteins/metabolism , Base Pairing , Base Sequence , HEK293 Cells , Humans , Methylation , Protein Binding , RNA, Double-Stranded/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Induced Silencing Complex/genetics , RNA-Induced Silencing Complex/metabolism
Small interfering RNAs (siRNAs) can be utilized not only as functional biological research tools but also as therapeutic agents. For the clinical use of siRNA as drugs, various chemical modifications have been used to improve the activity of siRNA drugs, and further chemical modifications are expected to improve the utility of siRNA therapeutics. As the 5' nucleobase of the guide strand affects the interaction between an siRNA and AGO2 and target cleavage activity, structural optimization of this specific position may be a useful strategy for improving siRNA activity. Here, using the in silico model of the complex between human AGO2 MID domain and nucleoside monophosphates, we screened and synthesized an original adenine-derived analog, 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), that fits better than the natural nucleotide bases into the MID domain of AGO2. Introduction of the 6-mCEPh-purine analog at the 5'-end of the siRNA guide strand significantly enhanced target knockdown activity in both cultured cell lines and in vivo animal models. Our findings can help expand strategies for rationally optimizing siRNA activity via chemical modifications of nucleotide bases.
Adenine/pharmacology , Argonaute Proteins/genetics , RNA Interference/drug effects , RNA, Double-Stranded/genetics , RNA, Small Interfering/agonists , RNA-Induced Silencing Complex/agonists , Adenine/analogs & derivatives , Adenine/chemical synthesis , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Animals , Apolipoprotein B-100/antagonists & inhibitors , Apolipoprotein B-100/blood , Apolipoprotein B-100/chemistry , Apolipoprotein B-100/genetics , Argonaute Proteins/metabolism , Base Pairing , Base Sequence , Binding Sites , Cholesterol/blood , HeLa Cells , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Male , Methylation , Mice , Mice, Knockout , Models, Molecular , Protein Binding , RNA, Double-Stranded/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Induced Silencing Complex/genetics , RNA-Induced Silencing Complex/metabolism , Uridine Monophosphate/chemistry , Uridine Monophosphate/metabolism
