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Activation of different split functionalities on re-association of RNA–DNA hybrids

Abstract

Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA–DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of ‘smart’ nucleic acid-based nanoparticles and switches for various biomedical applications.

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Figure 1: Activation of functionality by two RNA–DNA hybrids.
Figure 2: Fluorescent studies of RNA–DNA hybrid re-association in solution at 37 °C.
Figure 3: Re-association and localization of RNA–DNA hybrids in human breast cancer cells (MDA-MB-231) visualized by confocal fluorescence microscopy.
Figure 4: GFP knockdown assays for human breast cancer cells (MDA-MB-231/GFP) that stably express enhanced GFP (eGFP).
Figure 5: In vivo and ex vivo studies of RNA–DNA hybrids in a tumour xenograft mouse model.
Figure 6: HIV-1 expression and production is inhibited by siRNAs and recombined RNA–DNA hybrids.

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Acknowledgements

K.A.A. dedicates this work to T. Vinogradova and V. Vinogradov. The authors thank M.A. Dobrovolskaia from the Nanotechnology Characterization Laboratory for critical reading and helping to address some of the reviewers’ concerns. The authors also thank N. Patel, L. Riffle and J. Kalen in the Small Animal Imaging Program at the Frederick National Laboratory for Cancer Research for their guidance and support in animal imaging. The authors acknowledge help with ex vivo imaging from the Pathology Histology Laboratory of SAIC-Frederick at the Frederick National Laboratory for Cancer Research Thanks also go to C.J. Westlake at the Frederick National Laboratory for Cancer Research for providing plasmids expressing GFP-Rab5 or GFP-Rab7. The authors thank P.S. Steeg at the National Cancer Institute for providing GFP-expressing cells for in vivo experiments. This research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This work was funded, in whole or in part, by Federal funds from the Frederick National Laboratory for Cancer Research, NIH (contract no. HHSN261200800001E). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. This research was also supported by an NIH grant (no. R01GM-079604 to L.J.).

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K.A.A. and B.A.S. designed the project. K.A.A., M.V., B.A.S., A.N.M. and E.O.F. conceived and designed the experiments. K.A.A. performed all sequence designs and assemblies, Dicer assay experiments and human serum degradation studies. K.A.A. and M.V. performed all in vitro fluorescent studies and related cell transfections. K.A.A., M.V. and S.J.L. performed microscopy experiments and related analysis. R.B., M.V. and K.A.A. determined the kinetics. K.A.A., M.V., A.N.M. and A.E.M. carried out all silencing experiments. K.A.A. and M.V. contributed to in vivo and ex vivo studies and data analysis. E.H. provided material for in vivo studies. K.A.A., M.V., A.N.M., A.E.M. E.O.F., L.J., S.J.L. and B.A.S. analysed data. K.A.A., M.V., B.A.S., A.N.M. and E.O.F. co-wrote the paper.

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Correspondence to Bruce A. Shapiro.

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Afonin, K., Viard, M., Martins, A. et al. Activation of different split functionalities on re-association of RNA–DNA hybrids. Nature Nanotech 8, 296–304 (2013). https://doi.org/10.1038/nnano.2013.44

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