Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells
Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells
复制标题
DOI：
10.1261/rna.701307
复制
发表时间：
2007-10-01
期刊：
影响因子：
4.5
通讯作者：
Rhoads, Robert E.
中科院分区：
文献类型：
--
作者：
Grudzien-Nogalska, Ewa;Jemielity, Jacek;Rhoads, Robert E.
通过体外转录合成的加帽RNA已被广泛用于研究mRNA功能和代谢以及用于产生感兴趣的蛋白质。我们在此表征了最近合成的一系列具有改进性质的帽类似物，其在α-、β-或γ-磷酸部分中的非桥氧分别含有硫取代，m（2）（7，2 '-O）Gpp（S）PG、m（2）（7，2'-O）Gpp（S）pG和m（2）（7，2 '-O）Gp（S）ppG。新化合物还在m（7）Guo的2 '-O位置进行了修饰，使其成为抗逆转录帽类似物（ARCA），即，它们在体外转录过程中仅以正确的方向掺入。每种S-ARCA以两种非对映异构体形式（D1和D2）存在，可通过反相HPLC进行分离。mRNA降解的主要体内途径是通过焦磷酸酶Dcp 1/Dcp 2去除帽来启动的，焦磷酸酶Dcp 1/Dcp 2在α-磷酸和β-磷酸之间裂解。用m（2）（7，2 '-O）Gpp（S）pG（D2）封端的寡核苷酸在体外对重组人Dcp 2的水解完全耐受，而用m（2）（7，2'-O）Gpp（S）pG（D1）封端的寡核苷酸和m（2）（7，2 '-O）Gppp（S）G的两种异构体封端的寡核苷酸部分耐受。在培养的HC 11乳腺上皮细胞中，m（2）（7，2 '-O）Gpp（S）pG（D2）加帽的荧光素酶mRNA的t（1/2）为257 min，而m（7）Gp（3）G加帽的mRNA的t（1/2）为86 min。用m（2）（7，2 '-O）Gpp（S）pG（D1）和m（2）（7，2'-O）Gpp（S）pG（D2）加帽的荧光素酶mRNA在HC 11细胞中的翻译效率分别是用m（7）Gp（3）G加帽的荧光素酶mRNA的2.8倍和5.1倍。由于将更高的翻译效率与更长的mRNA的t（1/2）相结合而产生的更大的蛋白质产量应该有益于利用RNA转染的应用，例如蛋白质生产、抗癌免疫和基因治疗。
Capped RNAs synthesized by in vitro transcription have found wide utility for studying mRNA function and metabolism and for producing proteins of interest. We characterize here a recently synthesized series of cap analogs with improved properties that contain a sulfur substitution for a nonbridging oxygen in either the alpha-, beta-, or gamma- phosphate moieties, m(2)(7,2'-O)Gppp(S)G, m(2)(7,2'-O)Gpp(S)pG, and m(2)(7,2'-O)Gp(S)ppG, respectively. The new compounds were also modified at the 2'-O position of the m(7)Guo to make them anti-reverse cap analogs (ARCAs), i.e., they are incorporated exclusively in the correct orientation during in vitro transcription. Each of the S-ARCAs exists in two diastereoisomeric forms (D1 and D2) that can be resolved by reverse-phase HPLC. A major in vivo pathway for mRNA degradation is initiated by removal of the cap by the pyrophosphatase Dcp1/Dcp2, which cleaves between the alpha-and beta-phosphates. Oligonucleotides capped with m(2)(7,2'-O)Gpp(S)pG (D2) were completely resistant to hydrolysis by recombinant human Dcp2 in vitro, whereas those capped with m(2)(7,2'-O)Gpp(S)pG (D1) and both isomers of m(2)(7,2'-O)Gppp(S)G were partially resistant. Luciferase mRNA capped with m(2)(7,2'-O)Gpp(S)pG (D2) had a t(1/2) of 257 min in cultured HC11 mammary epithelial cells compared with 86 min for m(7)Gp(3)G-capped mRNA. Luciferase mRNAs capped with m(2)(7,2'-O)Gpp(S)pG (D1) and m(2)(7,2'-O)Gpp(S)pG (D2) were translated 2.8-fold and 5.1-fold, respectively, more efficiently in HC11 cells than those capped with m (7)Gp(3)G. The greater yield of protein due to combining higher translational efficiency with longer t(1/2) of mRNA should benefit applications that utilize RNA transfection such as protein production, anti-cancer immunization, and gene therapy.