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Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells

Nature Protocols volume 3, pages 1537–1549 (2008)Cite this article

Abstract

MicroRNAs (miRNAs), ∼22-nt RNAs that mediate post-transcriptional regulation of mRNAs in animals and plants, are a diverse class of regulatory genes whose specific biological functions are largely unknown. Here we detail a protocol to design and introduce into cultured Drosophila and human cells sequence-specific antisense oligonucleotides (ASOs) that block the function of individual miRNAs. Coupled with recent studies that catalog the miRNAs expressed in diverse cultured cells, our method offers a rapid (<1 week) approach to validate miRNA targets and to study the cellular functions of individual human and Drosophila miRNAs. ASO-based inactivation of miRNAs is faster and simpler than comparable genetic or 'sponge'-based approaches, for which extensive recombinant DNA manipulation is required. We present our ASO design principles and an optimized transfection protocol in which transfection efficiency of Drosophila Schneider 2 cells can approach 100%. Our 3′-cholesterol-modified ASOs have enhanced potency, allowing miRNA inhibition for at least 7 d from a single transfection.

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Figure 1: Model for ASO disruption of miRNA silencing.
Figure 2: Chemical structures of ASOs used to block miRNA function.
Figure 3: Design of miRNA sensor reporter target sites.
Figure 4: A sensitive reporter system for miR-277 silencing.
Figure 5: Efficient transfection requires optimal cell density.

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Acknowledgements

We thank Klaus Förstemann for constructing GFP reporters and stable S2 cell lines. This work was supported in part by grants from the National Institutes of Health to P.D.Z. (GM62862 and GM65236) and an NRSA MD/PhD pre-doctoral Fellowship from the National Institute on Aging to M.D.H. (F30AG030283).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, 01605, Massachusetts, USA

    Michael D Horwich & Phillip D Zamore

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  1. Michael D Horwich
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Correspondence to Phillip D Zamore.

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Competing interests

PDZ is a cofounder and scientific advisory board member of Alnylam Pharmaceuticals, Inc., and a scientific advisory board member of Regulus Therapeutics, LLC.

Supplementary information

Supplementary Fig. 1

Enhanced miRNA Inhibition with 3′ Cholesterol-Modified ASO. (PDF 154 kb)

Supplementary Fig. 2

Improved Detection of Synthetic let-7 and Endogenous miR-277 in the Presence of High Levels of Antagomir ASO using an LNA Northern Probe. (PDF 1918 kb)

Supplementary Fig. 3

Optimization of ASO Transfection. (PDF 358 kb)

Supplementary Data 1

Optimization and Comparison of 3’ Cholesterol-Modified ASOs. (PDF 75 kb)

Supplementary Data 2

Enhanced Detection of miRNA:ASO Hybrids with LNA Northern Probe. (PDF 51 kb)

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Horwich, M., Zamore, P. Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells. Nat Protoc 3, 1537–1549 (2008). https://doi.org/10.1038/nprot.2008.145

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