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Gene silencing by synthetic U1 Adaptors

Nature Biotechnology volume 27, pages 257–263 (2009)Cite this article

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Abstract

We describe a gene silencing method that employs a mechanism of action distinct from those of antisense and RNA interference. U1 Adaptors are bifunctional oligonucleotides with a 'target domain' complementary to a site in the target gene's terminal exon and a 'U1 domain' that binds to the U1 small nuclear RNA component of the U1 small nuclear ribonucleoprotein (U1 snRNP) splicing factor. Tethering of U1 snRNP to the target pre-mRNA inhibits poly(A)-tail addition, causing degradation of that RNA species in the nucleus. U1 Adaptors can inhibit both endogenous and reporter genes in a sequence-specific manner. Comparison of U1 Adaptors with small interfering RNA (siRNA) using a genome-wide microarray analysis indicates that U1 Adaptors have limited off-target effects and no detectable adverse effects on splicing. Further, targeting the same gene either with multiple U1 Adaptors or with a U1 Adaptor and siRNA strongly enhances gene silencing.

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Figure 1: The U1 Adaptor concept.
Figure 2: U1 Adaptor–mediated inhibition of reporter plasmid expression.
Figure 3: Effect on activity of changes in U1 domain length, location and composition.
Figure 4: Inhibition of endogenous RAF1.
Figure 5: Inhibition of the endogenous PCSK9 and enhanced inhibition with multiple Adaptors.
Figure 6: Co-transfection of U1 Adaptors and an siRNA enhances silencing.

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Change history

  • 20 February 2009

    In the version of this article initially published online, the numbers ten and 15 were reversed in the nucleotide composition on the second page, column 1, lines 13-14. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

This work was supported by National Institutes of Health grants GM057286 and CA119934 to S.I.G. and R43GM085863 to M.A.B. We thank members of the Gunderson lab, the Rutgers University/University of Medicine and Dentistry New Jersey RNA community and IDT corporation for advice and comments during the course of this work. We also thank Chris Jakubowski and Nick Zaphiros for helping to perform experiments shown in Supplementary Figs. 7, 8 and 11, and we thank Kim Lennox for help in editing the manuscript.

Author information

Authors and Affiliations

  1. Rutgers University, 604 Allison Road, Piscataway, 08854, New Jersey, USA

    Rafal Goraczniak & Samuel I Gunderson

  2. Integrated DNA Technologies, 1710 Commercial Park, Coralville, 52241, Iowa, USA

    Mark A Behlke

Authors
  1. Rafal Goraczniak
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  3. Samuel I Gunderson
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Contributions

S.I.G. and R.G. conceived the project and are co-inventors of the U1 Adaptor technology. R.G. performed the bulk of the wet bench experimental work, with some assistance from S.I.G. All authors contributed to experimental design, data interpretation and preparation of the manuscript. M.A.B. provided most of the U1 Adaptors employed and assisted with antisense and RNAi aspects of the project.

Corresponding author

Correspondence to Samuel I Gunderson.

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Competing interests

S.I.G. and R.G. are employed by Rutgers University and are co-inventors of the U1Adaptor Technology. Rutgers Univesity has submitted a PCT application on this invention and is the sole owner of the technology. Recently S.I.G. and R.G. started a company with the goal to further develop and ultimately to commercialize the U1 Adaptor technology. M.A.B. is employed by integrated DNA Technologies (IDT), which offers oligonucleotides for sale similar to some of the compounds described in the paper. IDT is not a publicly traded company and M.A.B. does not own any shares or equity in it.

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Figures 1–18, Data (PDF 4132 kb)

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Goraczniak, R., Behlke, M. & Gunderson, S. Gene silencing by synthetic U1 Adaptors. Nat Biotechnol 27, 257–263 (2009). https://doi.org/10.1038/nbt.1525

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