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Ca(2+) enrichment in culture medium potentiates effect of oligonucleotides
Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca(2+) enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleot...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627064/ https://www.ncbi.nlm.nih.gov/pubmed/26101258 http://dx.doi.org/10.1093/nar/gkv626 |
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author | Hori, Shin-ichiro Yamamoto, Tsuyoshi Waki, Reiko Wada, Shunsuke Wada, Fumito Noda, Mio Obika, Satoshi |
author_facet | Hori, Shin-ichiro Yamamoto, Tsuyoshi Waki, Reiko Wada, Shunsuke Wada, Fumito Noda, Mio Obika, Satoshi |
author_sort | Hori, Shin-ichiro |
collection | PubMed |
description | Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca(2+) enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ∼100 nm in size are found in Ca(2+)-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory. |
format | Online Article Text |
id | pubmed-4627064 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-46270642015-11-13 Ca(2+) enrichment in culture medium potentiates effect of oligonucleotides Hori, Shin-ichiro Yamamoto, Tsuyoshi Waki, Reiko Wada, Shunsuke Wada, Fumito Noda, Mio Obika, Satoshi Nucleic Acids Res Methods Online Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca(2+) enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ∼100 nm in size are found in Ca(2+)-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory. Oxford University Press 2015-10-30 2015-06-22 /pmc/articles/PMC4627064/ /pubmed/26101258 http://dx.doi.org/10.1093/nar/gkv626 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Hori, Shin-ichiro Yamamoto, Tsuyoshi Waki, Reiko Wada, Shunsuke Wada, Fumito Noda, Mio Obika, Satoshi Ca(2+) enrichment in culture medium potentiates effect of oligonucleotides |
title | Ca(2+) enrichment in culture medium potentiates effect of oligonucleotides |
title_full | Ca(2+) enrichment in culture medium potentiates effect of oligonucleotides |
title_fullStr | Ca(2+) enrichment in culture medium potentiates effect of oligonucleotides |
title_full_unstemmed | Ca(2+) enrichment in culture medium potentiates effect of oligonucleotides |
title_short | Ca(2+) enrichment in culture medium potentiates effect of oligonucleotides |
title_sort | ca(2+) enrichment in culture medium potentiates effect of oligonucleotides |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627064/ https://www.ncbi.nlm.nih.gov/pubmed/26101258 http://dx.doi.org/10.1093/nar/gkv626 |
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