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Synthesis of (18)O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase s...

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Autores principales: Hamasaki, Tomohiro, Matsumoto, Takahiro, Sakamoto, Naoya, Shimahara, Akiko, Kato, Shiori, Yoshitake, Ayumi, Utsunomiya, Ayumi, Yurimoto, Hisayoshi, Gabazza, Esteban C., Ohgi, Tadaaki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695515/
https://www.ncbi.nlm.nih.gov/pubmed/23632164
http://dx.doi.org/10.1093/nar/gkt344
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author Hamasaki, Tomohiro
Matsumoto, Takahiro
Sakamoto, Naoya
Shimahara, Akiko
Kato, Shiori
Yoshitake, Ayumi
Utsunomiya, Ayumi
Yurimoto, Hisayoshi
Gabazza, Esteban C.
Ohgi, Tadaaki
author_facet Hamasaki, Tomohiro
Matsumoto, Takahiro
Sakamoto, Naoya
Shimahara, Akiko
Kato, Shiori
Yoshitake, Ayumi
Utsunomiya, Ayumi
Yurimoto, Hisayoshi
Gabazza, Esteban C.
Ohgi, Tadaaki
author_sort Hamasaki, Tomohiro
collection PubMed
description Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of (18)O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging (18)O atom into the phosphate group during the oxidation step of the synthetic cycle by using (18)O water as the oxygen donor. The (18)O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The (18)O/(16)O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of (18)O-labeled RNA, and this technique was used to determine the blood concentration of (18)O-labeled RNA after administration to mice. (18)O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.
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spelling pubmed-36955152013-06-28 Synthesis of (18)O-labeled RNA for application to kinetic studies and imaging Hamasaki, Tomohiro Matsumoto, Takahiro Sakamoto, Naoya Shimahara, Akiko Kato, Shiori Yoshitake, Ayumi Utsunomiya, Ayumi Yurimoto, Hisayoshi Gabazza, Esteban C. Ohgi, Tadaaki Nucleic Acids Res Methods Online Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of (18)O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging (18)O atom into the phosphate group during the oxidation step of the synthetic cycle by using (18)O water as the oxygen donor. The (18)O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The (18)O/(16)O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of (18)O-labeled RNA, and this technique was used to determine the blood concentration of (18)O-labeled RNA after administration to mice. (18)O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies. Oxford University Press 2013-07 2013-04-30 /pmc/articles/PMC3695515/ /pubmed/23632164 http://dx.doi.org/10.1093/nar/gkt344 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Hamasaki, Tomohiro
Matsumoto, Takahiro
Sakamoto, Naoya
Shimahara, Akiko
Kato, Shiori
Yoshitake, Ayumi
Utsunomiya, Ayumi
Yurimoto, Hisayoshi
Gabazza, Esteban C.
Ohgi, Tadaaki
Synthesis of (18)O-labeled RNA for application to kinetic studies and imaging
title Synthesis of (18)O-labeled RNA for application to kinetic studies and imaging
title_full Synthesis of (18)O-labeled RNA for application to kinetic studies and imaging
title_fullStr Synthesis of (18)O-labeled RNA for application to kinetic studies and imaging
title_full_unstemmed Synthesis of (18)O-labeled RNA for application to kinetic studies and imaging
title_short Synthesis of (18)O-labeled RNA for application to kinetic studies and imaging
title_sort synthesis of (18)o-labeled rna for application to kinetic studies and imaging
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695515/
https://www.ncbi.nlm.nih.gov/pubmed/23632164
http://dx.doi.org/10.1093/nar/gkt344
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