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Lipophilic Conjugates for Carrier-Free Delivery of RNA Importable into Human Mitochondria

Part of the Methods in Molecular Biology book series (MIMB,volume 2277)

Abstract

Defects in human mitochondrial genome can cause a wide range of clinical disorders that still do not have efficient therapies. The natural pathway of small noncoding RNA import can be exploited to address therapeutic RNAs into the mitochondria. To create an approach of carrier-free targeting of RNA into living human cells, we designed conjugates containing a cholesterol residue and developed the protocols of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable pH-triggered hydrazone bond. The biodegradable conjugates of importable RNA with cholesterol can be internalized by cells in a carrier-free manner; RNA can then be released in the late endosomes due to a change in pH and partially targeted into mitochondria. Here we provide detailed protocols for solid-phase and “in solution” chemical synthesis of oligoribonucleotides conjugated to a cholesterol residue through a hydrazone bond. We describe the optimization of the carrier-free cell transfection with these conjugated RNA molecules and methods for evaluating the cellular and mitochondrial uptake of lipophilic conjugates.

Key words

  • RNA therapeutics
  • Carrier-free cell delivery
  • Synthesis of lipophilic conjugates
  • Mammalian cells transfection
  • Fluorescent microscopy

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  • DOI: 10.1007/978-1-0716-1270-5_4
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Acknowledgements

The authors thank the Center of Chemical Investigations of N.N.Vorozhtsov, Novosibirsk Institute of Organic Chemistry SB RAS for NMR analysis.

This work was funded by Strasbourg University, RFBR and CNRS (joint project PRC CNRS/RFBR mito TARN 20-54-15005), French National Research Agency (ANR) through the Programme d’Investissement d’Avenir under contract ANR-17-EURE-0023 and Labex MitoCross, and partly supported by the Russian state-funded budget project of ICBFM SB RAS # 0245-2021-0007.

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Correspondence to Nina Entelis .

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Dovydenko, I., Meschaninova, M., Heckel, AM., Tarassov, I., Venyaminova, A., Entelis, N. (2021). Lipophilic Conjugates for Carrier-Free Delivery of RNA Importable into Human Mitochondria. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 2277. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1270-5_4

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  • DOI: https://doi.org/10.1007/978-1-0716-1270-5_4

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