Abstract
Knockdown of genes by RNA interference (RNAi) in vitro requires methods of transfection or transduction, both of which have limited impact in vivo. As a virus-free approach, we chemically coupled cell surface receptors internalizing antibodies to the short interfering RNA (siRNA) carrier peptide protamine using the bispecific cross-linker sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). First, protamine was conjugated amino-terminally to sulfo-SMCC, and then this conjugate was coupled via cysteine residues to the IgG backbone to carry siRNA. This complex can efficiently find, bind and internalize into receptor-positive cells in vitro and in vivo, which can be checked by flow cytometry, fluorescence microscopy and western blotting. This method obtains results similar to those of siRNA targeting molecules engineered by genetic fusions between receptor-binding and siRNA carrier units, with the advantage of using readily available purified proteins without the need for engineering, expression and purification of respective constructs. The procedure for coupling the complex takes ∼2 d, and the functional assays take ∼2 weeks.
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Acknowledgements
This work was supported by the Wilhelm Sander Stiftung grant nos. 2009.041.2 (to S.B. and C.M.-T.), 2014.054.1 (to W.E.B. and S.B.) and 2010.015.1 (to W.E.B.); by Deutsche Forschungsgemeinschaft, DFG EXC 1003 Cells in Motion, Cluster of Excellence (to W.E.B.); and by Innovative Medizinische Forschung (IMF) grant nos. 111418 (to S.B.) and 121314 (to N.B.) by the Medical Faculty, University of Münster.
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N.B., N.A. and S.B. wrote most of the protocol. N.B., N.A., L.T., F.B., C.R. and S.B. performed the experiments and analyzed the data. S.B., C.M.-T. and W.E.B. initially designed the protocol and supervised the study. All authors contributed to writing the paper.
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Bäumer, N., Appel, N., Terheyden, L. et al. Antibody-coupled siRNA as an efficient method for in vivo mRNA knockdown. Nat Protoc 11, 22–36 (2016). https://doi.org/10.1038/nprot.2015.137
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DOI: https://doi.org/10.1038/nprot.2015.137
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