An Influence of Modification with Phosphoryl Guanidine Combined with a 2′-O-Methyl or 2′-Fluoro Group on the Small-Interfering-RNA Effect

Ilya S. Dovydenko,Inna A. Pyshnaya,Anna V. Epanchitseva,Anna S. Pavlova,Elena I. Ryabchikova,Maxim S. Kupryushkin,Kristina I. Yakovleva,Dmitrii V. Pyshnyi

doi:10.3390/ijms22189784

Abstract

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2′-O-methyl (2′-OMe) or PG/2′-fluoro (2′-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.

Highlights

RNA interference (RNAi) is a natural mechanism for regulating the expression of protein-coding genes in most of the eukaryotic cells
Our results suggested that the introduction of phosphoryl guanidine (PG) groups substantially protects a duplex at the 30 end of the antisense chain and can cause RNAse antisense chain chain (As) to change its position of interaction with the Small interfering RNA (siRNA)
Our findings clarify the feasibility of using of PG groups for siRNA modification

Summary

Introduction

RNA interference (RNAi) is a natural mechanism for regulating the expression of protein-coding genes in most of the eukaryotic cells. The involvement of small interfering RNAs (siRNAs) in the RNAi pathway leads to translation inhibition due to the degradation of a homologous mRNA target [2]. Th improve siRNA properties by chemical modifications has aroused much interes researchers, and a number of different modifications have been proposed. One type of m mines the localization of chemical modifications that can be placed in an internucleotide tion is notribose sufficient the properties a particular siRNA, isaccording to phosphate, moiety,toorimprove nucleoside [10]. As a rule,of one type of modification not on patisiran and the givosiran thesiRNA, development new chemical modifica sufficient to improve properties[9]. These functionalized siRNAs were studied regarding thermal stability, R the ability to induce RNAi in cultured mammalian cells in com

Design

Duplex Thermostability

Cleavage

Cleavage site determination in siRNAs

The Activity of Modifed siRNA In Vitro

Relative

Reagents

Oligonucleotide Synthesis

UV-Melting Experiments

RNAse A Degradation Assays

Findings

Cell Culture and Transfection