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dc.contributor.authorLy, Socheata
dc.contributor.authorNavaroli, Deanna M.
dc.contributor.authorDidiot, Marie-Claire
dc.contributor.authorCardia, James
dc.contributor.authorPandarinathan, Lakshmipathi
dc.contributor.authorAlterman, Julia F.
dc.contributor.authorFogarty, Kevin E.
dc.contributor.authorStandley, Clive
dc.contributor.authorLifshitz, Lawrence
dc.contributor.authorBellve, Karl D.
dc.contributor.authorProt, Matthieu
dc.contributor.authorEcheverria, Dimas
dc.contributor.authorCorvera, Silvia
dc.contributor.authorKhvorova, Anastasia
dc.date2022-08-11T08:09:46.000
dc.date.accessioned2022-08-23T16:42:51Z
dc.date.available2022-08-23T16:42:51Z
dc.date.issued2017-01-09
dc.date.submitted2017-03-27
dc.identifier.citationNucleic Acids Res. 2017 Jan 9;45(1):15-25. doi: 10.1093/nar/gkw1005. Epub 2016 Nov 28. <a href="https://doi.org/10.1093/nar/gkw1005">Link to article on publisher's site</a>
dc.identifier.issn0305-1048 (Linking)
dc.identifier.doi10.1093/nar/gkw1005
dc.identifier.pmid27899655
dc.identifier.urihttp://hdl.handle.net/20.500.14038/40176
dc.description.abstractsiRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs(R)) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe approximately 25% of sd-rxRNA co-localizing with EGF and < 5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=27899655&dopt=Abstract">Link to Article in PubMed</a>
dc.rightsCopyright © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectBiochemistry, Biophysics, and Structural Biology
dc.titleVisualization of self-delivering hydrophobically modified siRNA cellular internalization
dc.typeJournal Article
dc.source.journaltitleNucleic acids research
dc.source.volume45
dc.source.issue1
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=3979&amp;context=oapubs&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/2974
dc.identifier.contextkey9928023
refterms.dateFOA2022-08-23T16:42:51Z
html.description.abstract<p>siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs(R)) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe approximately 25% of sd-rxRNA co-localizing with EGF and < 5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.</p>
dc.identifier.submissionpathoapubs/2974
dc.contributor.departmentUMass Metabolic Network
dc.contributor.departmentRNA Therapeutics Institute
dc.contributor.departmentProgram in Molecular Medicine
dc.source.pages15-25


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Copyright © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
Except where otherwise noted, this item's license is described as Copyright © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.