Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety
Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety
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将稳定同位素掺入碱基部分的核苷的酶促合成和 RNA 干扰
DOI：
10.1016/j.bmc.2015.09.011
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发表时间：
2015
影响因子：
3.5
通讯作者：
Kenji Fukuda
中科院分区：
文献类型：
--
作者：
Akihiko Hatano;Mitsuya Shiraishi;Nanae Terado;Atsuhiro Tanabe;Kenji Fukuda
胸苷磷酸化酶用于催化掺入稳定同位素的胸苷（或甲基尿苷）和尿嘧啶转化为脱氧尿苷（或尿苷），尿嘧啶碱基掺入稳定同位素。这些碱交换反应以高转化率（75-96%）进行，并且分离产率也很好（64-87%）。所有合成的化合物的质量与理论分子量相同，通过EIMS。13 C NMR谱显示合成的化合物中13 C和15 N之间的自旋-自旋耦合，并且信号分裂，进一步证明了同位素掺入到化合物中。还研究了这种siRNA与掺入稳定同位素的尿苷的RNA干扰效果。25 mer siRNA对MARCKS蛋白具有强的敲低作用。掺入稳定同位素的尿苷部分的插入位置和数目引入siRNA中对靶蛋白的沉默没有影响。这种将稳定同位素掺入RNA和DNA中的方法有可能在细胞中起到化学良性示踪剂的作用。
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.13C NMR spectra showed spin–spin coupling between13C and15N 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.