Abstract
The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.
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Acknowledgements
The authors wish to thank A. Koustrup, H. Brostrøm, A. Konge, J.J. Jørgensen, M. R. Møller, L. Bang and M. Meldgaard for excellent technical assistance, and A. Lund and T. Bou-Kheir for reagents. This study was supported by grants from the Danish National Advanced Technology Foundation and the Danish Medical Research Council to S.K.
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S.O., C.O.d.S., A.P., M.H., O.B., C.R., C.F. and E.M.S. performed experiments and contributed data. H.F.H. performed synthesis of oligonucleotides. S.O., C.O.d.S., A.P., M.H., M.L., J.S., T.K., D.P., G.J.H. and S.K. designed experiments and discussed the data. S.K. supervised the study and wrote the manuscript together with S.O. and with input from other authors.
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S.O., O.B., A.P., M.H., M.L., J.S., E.M.S., H.F.H., T.K. and S.K. are employees of Santaris Pharma, a clinical stage biopharmaceutical company that develops RNA-based therapeutics.
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Obad, S., dos Santos, C., Petri, A. et al. Silencing of microRNA families by seed-targeting tiny LNAs. Nat Genet 43, 371–378 (2011). https://doi.org/10.1038/ng.786
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DOI: https://doi.org/10.1038/ng.786
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