Gene silencing in mammalian cells by preformed small RNA duplexes

doi:10.1016/S0006-291X(02)00736-2

Biochemical and Biophysical Research Communications

Volume 295, Issue 3, 19 July 2002, Pages 744-748

https://doi.org/10.1016/S0006-291X(02)00736-2 Get rights and content

Abstract

Small interfering RNAs (siRNAs) mediate RNA interference (RNAi), a process in which target mRNAs are degraded. Here, we have investigated the efficacy of preformed siRNAs to modulate the expression of protein kinase Cα (PKCα) and green fluorescent protein (GFP) in mammalian cells. We show that specific inhibition of PKCα and GFP can be achieved by in vitro transcribed siRNAs. Interestingly, a transcript harboring two self-complementary siRNAs interrupted by a single-stranded loop region inhibited both PKCα and GFP gene expression. These results suggest that the long transcript is processed by single-stranded ribonucleases and/or other proteins into two functional siRNAs. Incubation of the in vitro transcribed bispecific siRNA with protein extracts from HEK 293T cells yielded RNA duplexes similar to the synthetic single siRNA. Taken together, the present data indicate that in vitro transcribed siRNA can be useful for silencing gene expression. Additionally, bi- and perhaps poly-siRNAs may be expressed and processed in mammalian cells.

Section snippets

Materials and methods

Chemically synthetic siRNAs against the protein kinase α (PKCα) and oligonucleotides were synthesized in an automatic synthesizer (EUROGENTEC, Belgium). In vitro transcribed siRNAs were synthesized from DNA template oligonucleotides using the T7 RNA polymerase as described by Milligan and Uhlenbeck [6]. For long double-stranded transcripts, the overlapping strategy was used [7]. After transcription, RNAs were purified by 15% polyacrylamide gels containing 7 M urea and stored at −70 °C until use.

Inhibition of PKCα gene expression by chemically and in vitro transcribed siRNAs

Previously, we have reported that active RNA ribozymes can be generated by an in vitro transcription strategy [8]. In this study, we have investigated whether in vitro made 21-nt siRNAs would function like their chemically synthetic counterparts [5]. Because the first nucleotide incorporated into the RNA by T7 RNA polymerase is a guanosine [6], in vitro transcribed siRNAs must start with at least one guanosine. We designed various gene-specific siRNAs targeting the protein kinase Cα and GFP (

Discussion

The ability of siRNA to interfere with gene expression in a specific manner is the basis for the therapeutic potential of these molecules especially in diseases where the production of aberrant proteins as well as the overexpression of normal proteins is a predominant feature. In the present study, we show that PKCα and GFP gene expression can be silenced by in vitro transcribed and chemically made siRNAs in a sequence-specific manner. Importantly, we show that long double-stranded siRNA with

Acknowledgements

This research was supported by the Norwegian Cancer Society. We thank Drs. Anne Dybwad and Øyvind Melien for their comments on the manuscript.

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^☆: Abbreviations: siRNA, small interfering RNA; GFP, green fluorescent protein; PKC, protein kinase C; RNAi, RNA interference.

View full text

Gene silencing in mammalian cells by preformed small RNA duplexes☆

Abstract

Section snippets

Materials and methods

Inhibition of PKCα gene expression by chemically and in vitro transcribed siRNAs

Discussion

Acknowledgements

Methods Enzymol.

J. Mol. Biol.

Nucleic acid enzymes as a novel generation of anti-gene agents

Curr. Mol. Med.

Catalytic nucleic acids: from lab to applications

Pharmacol. Rev.

RNA interference

Genes Dev.

A dsRNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis

Proc. Natl. Acad. Sci. USA

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Nature