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Synthetic oligonucleotides recruit ILF2/3 to RNA transcripts to modulate splicing

Nature Chemical Biology volume 8, pages 555–561 (2012)Cite this article

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Abstract

We describe a new technology for recruiting specific proteins to RNA through selective recognition of heteroduplexes formed with chemically modified antisense oligonucleotides (ASOs). Typically, ASOs function by hybridizing to their RNA targets and blocking the binding of single-stranded RNA–binding proteins. Unexpectedly, we found that ASOs with 2′-deoxy-2′-fluoro (2′-F) nucleotides, but not with other 2′ chemical modifications, have an additional property: they form heteroduplexes with RNA that are specifically recognized by the interleukin enhancer-binding factor 2 and 3 complex (ILF2/3). 2′-F ASO–directed recruitment of ILF2/3 to RNA can be harnessed to control gene expression by modulating alternative splicing of target transcripts. ILF2/3 recruitment to precursor mRNA near an exon results in omission of the exon from the mature mRNA, both in cell culture and in mice. We discuss the possibility of using chemically engineered ASOs that recruit specific proteins to modulate gene expression for therapeutic intervention.

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Figure 1: Modulation of splicing in cell culture as a function of ASO chemistry.
Figure 2: Identification of proteins that are recruited to a 2′-F ASO–RNA duplex.
Figure 3: 2′-F ASO–directed exon skipping is mediated by ILF2/3.
Figure 4: Structural determinants for ILF2/3 binding to ASO–RNA duplexes.
Figure 5: Effect of 2′-F mixmer ASOs on exon skipping.
Figure 6: Modulation of splicing in mice as a function of ASO chemistry.
Figure 7: Scheme showing the proposed mechanism for ASO chemistry–dependent modulation of SMN2 exon 7 alternative splicing.

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Acknowledgements

We thank A. Berdeja for Tm measurements and E. Swayze, W. Lima and D. Corey for helpful discussions.

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Authors and Affiliations

  1. Isis Pharmaceuticals, Carlsbad, California, USA

    Frank Rigo, Seung J Chun, Thazha P Prakash & C Frank Bennett

  2. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Yimin Hua & Adrian R Krainer

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  1. Frank Rigo
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Contributions

F.R., Y.H., T.P.P., A.R.K. and C.F.B. conceived of and designed the experiments. F.R. and S.J.C. performed the experiments. F.R. and C.F.B. analyzed the data. T.P.P. synthesized the ASOs. F.R. and C.F.B. wrote the paper.

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Correspondence to C Frank Bennett.

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Competing interests

F.R., S.J.C., T.P.P. and C.F.B. are employees of ISIS Pharmaceuticals. A.R.K. serves on the scientific advisory board of two nonprofit spinal muscular atrophy foundations and is a consultant for ISIS Pharmaceuticals.

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Rigo, F., Hua, Y., Chun, S. et al. Synthetic oligonucleotides recruit ILF2/3 to RNA transcripts to modulate splicing. Nat Chem Biol 8, 555–561 (2012). https://doi.org/10.1038/nchembio.939

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