Strategic Headquarters for Research and Development, BONAC Corporation, BIO Factory 4F, 4-1488 Aikawa, Kurume, Fukuoka 839-0861, Japan.
Nucleic Acids Res. 2013 Jul;41(12):e126. doi: 10.1093/nar/gkt344. Epub 2013 Apr 30.
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.
放射性同位素和荧光化合物常用于 RNA 标记，但由于辐射暴露的风险或共价连接的荧光团引起的不等效性，不适合用于 RNA 药物的临床研究。在这里，我们报告了一种实用的（18）O 标记 RNA 的亚磷酰胺固相合成方法，该方法避免了这些缺点，并展示了其在定量和成像方面的应用。该合成方法通过使用（18）O 水作为氧供体，在合成循环的氧化步骤中，将非桥接（18）O 原子引入到磷酸基团中。在 pH 3-8.5 范围内，RNA 中的（18）O 标记是稳定的，而通过圆二色性、熔点和 RNA 干扰活性，标记和未标记的短干扰 RNA 的理化和生物学性质是无法区分的。通过同位素比质谱测量的（18）O/（16）O 比值随（18）O 标记 RNA 浓度的增加呈线性增加，该技术用于测定（18）O 标记 RNA 给药后在小鼠血液中的浓度。用同位素显微镜观察转染到人类 A549 细胞中的（18）O 标记 RNA。在细胞核周围的细胞质中的焦点中观察到 RNA，推测对应于内体。这些方法可能对基础和药物研究中 RNA 的动力学和细胞定位研究有用。
