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Method for Confirming Cytoplasmic Delivery of RNA Aptamers

  • Protocol
SiRNA Delivery Methods

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1364))

Abstract

RNA aptamers are single-stranded RNA oligos that represent a powerful emerging technology with potential for treating numerous diseases. More recently, cell-targeted RNA aptamers have been developed for delivering RNA interference (RNAi) modulators (siRNAs and miRNAs) to specific diseased cells (e.g., cancer cells or HIV infected cells) in vitro and in vivo. However, despite initial promising reports, the broad application of this aptamer delivery technology awaits the development of methods that can verify and confirm delivery of aptamers to the cytoplasm of target cells where the RNAi machinery resides. We recently developed a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA). To assess cytoplasmic delivery, the aptamer was chemically conjugated to saporin, a ribosome inactivating protein toxin that is toxic to cells only when delivered to the cytoplasm (where it inhibits the ribosome) by a cell-targeting ligand (e.g., aptamer). Here, we describe the chemistry used to conjugate the aptamer to saporin and discuss a gel-based method to verify conjugation efficiency. We also detail an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity.

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Authors and Affiliations

  1. Department of Internal Medicine, University of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, 52242, USA

    David D. Dickey, Gregory S. Thomas, Justin P. Dassie & Paloma H. Giangrande Ph.D.

  2. Department of Radiation Oncology, University of Iowa, Iowa City, IA, 52242, USA

    Paloma H. Giangrande Ph.D.

Authors
  1. David D. Dickey
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  2. Gregory S. Thomas
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  3. Justin P. Dassie
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  4. Paloma H. Giangrande Ph.D.
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Corresponding author

Correspondence to Paloma H. Giangrande Ph.D. .

Editor information

Editors and Affiliations

  1. Beckman Research Institute of City of Hope, Duarte, California, USA

    Kato Shum

  2. Beckman Research Institute of City of Hope, Duarte, California, USA

    John Rossi

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Dickey, D.D., Thomas, G.S., Dassie, J.P., Giangrande, P.H. (2016). Method for Confirming Cytoplasmic Delivery of RNA Aptamers. In: Shum, K., Rossi, J. (eds) SiRNA Delivery Methods. Methods in Molecular Biology, vol 1364. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3112-5_17

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  • DOI: https://doi.org/10.1007/978-1-4939-3112-5_17

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3111-8

  • Online ISBN: 978-1-4939-3112-5

  • eBook Packages: Springer Protocols

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