Functional comparison of single- and double-stranded siRNAs in mammalian cells

https://doi.org/10.1016/j.bbrc.2004.02.100Get rights and content

Abstract

The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19–25 bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double- and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them.

Section snippets

Materials and methods

Generation of small interfering RNAs. Renilla luciferase mRNA (Accession No. AF025846) or CD46 mRNA (Accession No. XM_036622) were used as siRNA targets. siRNA target sequences in the two mRNA targets were selected partially by using the siRNA Target Finder and Design Tool (Ambion, USA), as described in our previous report [18]. The CD46-specific siRNAs were designed to target the conserved exon 6 and 9 in the CD46 isoforms [19]. Single-stranded antisense siRNAs and sense siRNAs were chemically

Duplex siRNAs were more potent RNAi triggers than single-stranded antisense siRNAs

We compared the functions of duplex and single-stranded siRNAs by targeting nine sites in the exogenous Renilla luciferase mRNA in HEK-293 cells. The duplex siRNAs were found to be more potent than the antisense siRNAs (Fig. 1A). The average of inhibitory effects generated by four most effective duplex siRNAs (targeting the sites RL82-100, RL375-393, RL501-519, and RL548-566) was 94%, while that generated by four most effective antisense siRNAs (targeting the sites RL82-100, RL479-497,

Discussion

There was a discrepancy in activities of single-stranded siRNAs in previous reports. Single-stranded antisense siRNAs were found to be inefficient, while duplex siRNAs were efficient, in most previous reports [10], [11], [12], [13]. In agreement with those findings, our data showed the priority of duplex siRNAs in knocking down exogenous luciferase and endogenous CD46 in two mammalian cell lines. Compared to those effective duplex siRNAs reported in the literature, documented effective

Acknowledgements

This research was supported by Microbiology and Tumor Biology Center (MTC), Karolinska Institute to Y.X., and Center for Genomics and Bioinformatics, KI and Pharmacia to Z.L. The authors wish to thank Lifu Hu and Xiangning Zhang for help with the TD-20/20 luminometer; and Birgitta Wester, for technical support in FACS, at MTC, Karolinska Institute.

References (22)

  • G. Sui et al.

    A DNA vector-based RNAi technology to suppress gene expression in mammalian cells

    Proc. Natl. Acad. Sci. USA

    (2002)
  • Cited by (0)

    View full text