Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland.
Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland.
Philos Trans R Soc Lond B Biol Sci. 2018 Nov 5;373(1762):20180167. doi: 10.1098/rstb.2018.0167.
The 5'-terminus of eukaryotic mRNAs comprises a 7-methylguanosine cap linked to the first transcribed nucleotide via a 5'-5' triphosphate bond. This cap structure facilitates numerous interactions with molecules participating in mRNA processing, turnover and RNA translation. Here, we report the synthesis and biochemical properties of a set of biotin-labelled cap analogues modified within the triphosphate bridge and increasing mRNA stability while retaining biological activity. Successful co-transcriptional incorporation of the cap analogues allowed for the quantification of cap-dependent translation efficiency, capping efficiency and the susceptibility to decapping by Dcp2. The utility of such cap-biotinylated RNAs as molecular tool was demonstrated by ultraviolet-cross-linking and affinity capture of protein-RNA complexes. In conclusion, RNAs labelled with biotin via the 5' cap structure can be applied to a variety of biological experiments based on biotin-avidin interaction or by means of biotin-specific antibodies, including protein affinity purification, pull-down assays, visualization, cellular delivery and many others.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.
真核生物 mRNAs 的 5' 端包含一个通过 5'-5' 三磷酸键连接到第一个转录核苷酸的 7-甲基鸟苷帽。这种帽结构促进了与参与 mRNA 加工、周转和 RNA 翻译的分子的许多相互作用。在这里，我们报告了一组在三磷酸桥内修饰的生物素标记帽类似物的合成和生化特性，这些类似物在保留生物活性的同时增加了 mRNA 的稳定性。这些帽类似物的成功转录共掺入允许定量测定帽依赖性翻译效率、加帽效率和 Dcp2 脱帽的敏感性。通过紫外线交联和蛋白质-RNA 复合物的亲和捕获，证明了这种带有生物素标记的帽 RNA 作为分子工具的实用性。总之，通过 5' 帽结构用生物素标记的 RNA 可应用于基于生物素-亲和素相互作用或生物素特异性抗体的各种生物学实验，包括蛋白质亲和纯化、下拉测定、可视化、细胞递送和许多其他实验。本文是主题为“5' 和 3' 修饰控制 RNA 降解”的特刊的一部分。
