title: Synthesis of ¹⁸O-labeled RNA for application to kinetic studies and imaging.
abstract: 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.
authors: Strategic Headquarters for Research and Development, BONAC Corporation, BIO Factory 4F, 4-1488 Aikawa, Kurume, Fukuoka 839-0861, Japan.
authors: Tomohiro
authors: T
authors: Hamasaki
authors: Takahiro
authors: T
authors: Matsumoto
authors: Naoya
authors: N
authors: Sakamoto
authors: Akiko
authors: A
authors: Shimahara
authors: Shiori
authors: S
authors: Kato
authors: Ayumi
authors: A
authors: Yoshitake
authors: Ayumi
authors: A
authors: Utsunomiya
authors: Hisayoshi
authors: H
authors: Yurimoto
authors: Esteban C
authors: EC
authors: Gabazza
authors: Tadaaki
authors: T
authors: Ohgi
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008853:Microscopy / Q000379:methods; D010103:Oxygen Isotopes; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000032:analysis / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D010103:Oxygen Isotopes; D034741:RNA, Small Interfering
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