title: Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety.
abstract: Thymidine phosphorylase was used to catalyze the conversion of thymidine (or methyluridine) and uracil incorporating stable isotopes to deoxyuridine (or uridine) with the uracil base incorporating the stable isotope. These base-exchange reactions proceeded with high conversion rates (75-96%), and the isolated yields were also good (64-87%). The masses of all synthetic compounds incorporating stable isotopes were identical to the theoretical molecular weights via EIMS. (13)C NMR spectra showed spin-spin coupling between (13)C and (15)N in the synthetic compounds, and the signals were split, further proving incorporation of the isotopes into the compounds. The RNA interference effects of this siRNA with uridine incorporating stable isotopes were also investigated. A 25mer siRNA had a strong knockdown effect on the MARCKS protein. The insertion position and number of uridine moieties incorporating stable isotopes introduced into the siRNA had no influence on the silencing of the target protein. This incorporation of stable isotopes into RNA and DNA has the potential to function as a chemically benign tracer in cells.
authors.affiliation: Department of Chemistry, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan. Electronic address: a-hatano@sic.shibaura-it.ac.jp.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Hatano
authors.affiliation: Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Mitsuya
authors.initials: M
authors.lastname: Shiraishi
authors.affiliation: Department of Chemistry, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan.
authors.forename: Nanae
authors.initials: N
authors.lastname: Terado
authors.affiliation: Department of Chemistry, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan.
authors.forename: Atsuhiro
authors.initials: A
authors.lastname: Tanabe
authors.affiliation: Taiyo Nippon Sanso Corp., 10 Okubo, Tsukuba-shi, Ibaragi 300-2611, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Fukuda
mesh_terms: D055162:Biocatalysis; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D013936:Thymidine / Q000737:chemistry / Q000378:metabolism*; D013939:Thymidine Phosphorylase / Q000378:metabolism*; D014498:Uracil / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D034741:RNA, Small Interfering; D014498:Uracil; D013939:Thymidine Phosphorylase; D013936:Thymidine
keywords: (13)C NMR; Nucleoside synthesis; Nucleosides incorporating stable isotopes; RNA interference; Thymidine phosphorylase