Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency
Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency
复制标题
DOI：
10.1261/rna.7380904
复制
发表时间：
2004-09-01
期刊：
影响因子：
4.5
通讯作者：
Rhoads, RE
中科院分区：
文献类型：
--
作者：
Grudzien, E;Stepinski, J;Rhoads, RE
在真核mRNA和snRNA的5'端的N7-甲基化鸟苷三磷酸帽的合成类似物在理解它们的剪接、细胞内转运、翻译和周转中发挥了重要作用。我们在此报道了一系列新的N7-苄基化二核苷四磷酸类似物，B（7）Gp（4）G，B（7）m（3-O）Gp（4）G和B（7）m（2）Gp（4）G，这些类似物扩展了我们对帽在翻译中的作用的认识。我们使用这些新的类似物，沿着10个先前合成的类似物，探索五个参数：与eIF 4 E的结合亲和力，在兔网织红细胞裂解物系统中帽依赖性翻译的抑制，在体外转录过程中掺入RNA的效率（%加帽），合成mRNA中类似物的方向（%正确方向），以及用类似物加帽的mRNA的体外翻译效率。13个帽类似物在第一（远端）和第二（近端）鸟嘌呤部分、第一和第二核糖部分以及磷酸残基的数目的修饰上不同。其中包括天然存在的cap m（3）（2，2，7）Gp（3）G的类似物。这些化合物对eIF 4 E的亲和力变化61倍，对帽依赖性翻译的抑制变化146倍，加帽%变化1.4倍，正确方向%变化5.6倍。最具刺激性的类似物与未加帽的RNA相比增强翻译44倍。用B（7）m（2）Gp（4）G、m（7）Gp（3）m（7）G、B（7）m（3 '-O）Gp（3）G和m（7）Gp（4）m（7）G加帽的mRNA的翻译效率分别比用m（7）Gp（3）G加帽的mRNA高2.5倍、2.6倍、2.8倍和3.1倍。相对翻译效率通常可以用对eIF 4 E的帽亲和力、%加帽和%正确取向来解释。所有五个参数的测量提供了对有助于翻译效率的因素的深入了解。
Synthetic analogs of the N7-methylated guanosine triphosphate cap at the 5' end of eukaryotic mRNAs and snRNAs have played an important role in understanding their splicing, intracellular transport, translation, and turnover. We report here a new series of N7-benzylated dinucleoside tetraphosphate analogs, b(7)Gp(4)G, b(7)m(3-O)Gp(4)G, and b(7)m(2)Gp(4)G, that extend our knowledge of the role of the cap in translation. We used these novel analogs, along with 10 previously synthesized analogs, to explore five parameters: binding affinity to eIF4E, inhibition of cap-dependent translation in a rabbit reticulocyte lysate system, efficiency of incorporation into RNAs during in vitro transcription (% capping), orientation of the analog in the synthetic mRNA (% correct orientation), and in vitro translational efficiency of mRNAs capped with the analog. The 13 cap analogs differed in modifications of the first (distal) and second (proximal) guanine moieties, the first and second ribose moieties, and the number of phosphate residues. Among these were analogs of the naturally occurring cap m(3)(2,2,7)Gp(3)G. These compounds varied by 61-fold in affinity for eIF4E, 146-fold in inhibition of cap-dependent translation, 1.4-fold in % capping, and 5.6-fold in % correct orientation. The most stimulatory analog enhanced translation 44-fold compared with uncapped RNA. mRNAs capped with b(7)m(2)Gp(4)G, m(7)Gp(3)m(7)G, b(7)m(3'-O)Gp(3)G, and m(7)Gp(4)m(7)G were translated 2.5-, 2.6-, 2.8-, and 3.1-fold more efficiently than mRNAs capped with m(7)Gp(3)G, respectively. Relative translational efficiencies could generally be explained in terms of cap affinity for eIF4E, % capping, and % correct orientation. The measurement of all five parameters provides insight into factors that contribute to translational efficiency.