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概述
牛痘病毒加帽体系利用牛痘病毒加帽酶及相关组分，将 7-甲基鸟苷帽结构（m7Gppp，Cap0）加到 RNA 的 5´ 末端。在真核生物中，该结构与 mRNA 的稳定、转运和翻译密切相关。使用酶促反应为 RNA 加帽是一种简单有效的方法，且帽结构与天然Cap 0 结构完全一致，能显著改善用于体外转录、转染和显微注射的 RNA 的稳定性和翻译能力。这种酶由两个亚基（D1 和D12）组成 ，D1 亚基执行 RNA 三磷酸酶和鸟苷转移酶的功能，D12 亚基执行鸟嘌呤甲基转移酶的功能，它们对于添加一个完整的 Cap 0 结构 m7Gppp5´N 都是必须的。
图1. 牛痘病毒加帽的结构和作用机理。a. 牛痘病毒加帽酶和 GTP（红色）、SAH（玫红）的共结晶结构 (PDB 4CKB) 。该酶由 D1 和 D12 两个亚基组成，兼具RNA 三磷酸酶（蓝色）、鸟苷转移酶（橙色）和鸟嘌呤甲基转移酶（米黄色）的功能。本图引自 Ramanathan, A., Robb, G. B., & Chan, S. H. (2016). mRNA capping: biological functions and applications. Nucleic Acids Res 44(16), 7511–7526. b. mRNA 的帽结构是由一个 7-甲基鸟苷通过一个5′–5′三磷酸酯桥连接到mRNA 链的5′核苷上组成。Cap-0 结构由相邻的 RNA 链通过三种酶的依次反应形成。进一步形成 cap-1 结构需要2’-O-甲基转移酶的参与，该修饰可以降低 RNA 在体内引起的细胞先天免疫反应。本图引自 Decroly, E., Ferron, F., Lescar, J. et al. (2012). Conventional and unconventional mechanisms for capping viral mRNA. Nat Rev Microbiol 10, 51–65。
本体系可用于 IVT RNA 的加帽反应，使加帽反应在 1h 之内完成，效率接近 100%，并且保证正确的方向。
产品用途
体内或体外翻译前 mRNA的加帽和 mRNA的 5’末端标记。
注意事项
SAM：需事先计算好 SAM 的用量，在反应开始前把分装的 32 mM 的储备液稀释成 2 mM 的工作液。SAM 在 pH 7-8，37°C条件下不稳定，需要在反应开始之前新鲜配置。为避免 SAM 降解，该工作液需要保存于冰上。
RNA：在加帽体系使用之前，应将体外转录反应产生的 RNA 进行纯化并溶解于 RNase-free 水中，不能将 RNA 溶解在 EDTA溶液或其他盐溶液中。
RNA 二级结构：一些 RNA 转录本可能形成稳定的二级结构（同源二聚体和发卡结构），如二级结构发生在转录本的 5'端会影响加帽效率。在与加帽酶反应之前加热 RNA 可以去除转录产物 5´端的二级结构，如果转录产物的 5´端结构复杂，可以把加热时间延长至 10min，加帽反应时间可延长至 60min。
Cap 0-和 Cap 1-mRNA：Cap 0-和 Cap 1-mRNA 之间的差异在 RNA 5'端紧邻帽结构的第一位核苷酸（N1）是否具有 2'-O-甲基。在高等真核细胞中，该甲基化是天然加帽过程的一部分，Cap 1 结构提高了 mRNA 的翻译效率。
Poly(A) 尾：如果加帽的 RNA 需要添加 3'-poly(A)尾巴，可以使用E. coli Poly(A) Polymerase进行加尾。加帽和加尾后的 RNA 需在进行 RNA 转染实验前做纯化处理。
RNase Inhibitor：在配置反应体系时，可以加入 0.5 µl RNase 抑制剂，同时去掉等体积的 RNase-free 水。
RNA5’端标记：在用于 RNA 5´端标记时，GTP 储备液应稀释到 mRNA 浓度的 1-3 倍。
The vaccinia virus capping system uses the vaccinia virus capping enzyme and related components to add the 7-methylguanosine cap structure (m7Gppp, Cap0) to the 5´ end of the RNA. In eukaryotes, this structure is closely related to the stability, transport and translation of mRNA. Using an enzymatic reaction to cap RNA is a simple and effective method, and the cap structure is exactly the same as the natural Cap 0 structure, which can significantly improve the stability and translation ability of RNA used for in vitro transcription, transfection, and microinjection. This enzyme is composed of two subunits (D1 and D12). The D1 subunit performs the functions of RNA triphosphatase and guanosine transferase, and the D12 subunit performs the function of guanine methyltransferase. They are essential for adding a complete The Cap 0 structure m7Gppp5´N is required.
Figure 1. The structure and mechanism of vaccinia virus capping. a. Co-crystal structure of vaccinia virus capping enzyme, GTP (red) and SAH (rose red) (PDB 4CKB). The enzyme is composed of two subunits, D1 and D12, and has the functions of RNA triphosphatase (blue), guanosine transferase (orange) and guanine methyltransferase (beige). This figure is quoted from Ramanathan, A., Robb, GB, & Chan, SH (2016). mRNA capping: biological functions and applications. Nucleic Acids Res 44(16), 7511–7526. b. The cap structure of mRNA consists of a 7-Methylguanosine is composed of a 5'-5' triphosphate bridge connected to the 5'nucleoside of the mRNA chain. The Cap-0 structure is formed by the sequential reaction of three adjacent RNA strands. The further formation of cap-1 structure requires the participation of 2'-O-methyltransferase, which can reduce the cellular innate immune response caused by RNA in vivo. This figure is quoted from Decroly, E., Ferron, F., Lescar, J. et al. (2012). Conventional and unconventional mechanisms for capping viral mRNA. Nat Rev Microbiol 10, 51–65.
This system can be used for the capping reaction of IVT RNA, so that the capping reaction is completed within 1 hour, the efficiency is close to 100%, and the correct direction is guaranteed.
Product Usage
In vivo or in vitro pre-translational capping of mRNA and 5'end labeling of mRNA.
Precautions
SAM: It is necessary to calculate the amount of SAM in advance, and dilute the aliquoted 32 mM stock solution to 2 mM working solution before the reaction starts. SAM is unstable at pH 7-8 and 37°C and needs to be freshly prepared before the reaction starts. To avoid degradation of SAM, this working solution needs to be stored on ice.
RNA: Before using the capping system, the RNA produced by the in vitro transcription reaction should be purified and dissolved in RNase-free water. The RNA should not be dissolved in EDTA solution or other salt solutions.
RNA secondary structure: Some RNA transcripts may form stable secondary structures (homodimers and hairpin structures). For example, the secondary structure occurring at the 5'end of the transcript will affect the capping efficiency. Heating RNA before reacting with the capping enzyme can remove the secondary structure at the 5´ end of the transcription product. If the 5´ end of the transcription product has a complex structure, the heating time can be extended to 10 minutes, and the capping reaction time can be extended to 60 minutes.
Cap 0- and Cap 1-mRNA: The difference between Cap 0- and Cap 1-mRNA is whether the first nucleotide (N1) next to the cap structure at the 5'end of RNA has a 2'-O-methyl group. In higher eukaryotic cells, this methylation is part of the natural capping process, and the Cap 1 structure improves the translation efficiency of mRNA.
Poly(A) tail: If the capped RNA needs to be added with 3'-poly(A) tail, E. coli Poly(A) Polymerase can be used for tailing. The capped and tailed RNA needs to be purified before the RNA transfection experiment.
RNase Inhibitor: When configuring the reaction system, you can add 0.5 µl of RNase inhibitor and remove an equal volume of RNase-free water.
RNA 5'end labeling: When used for RNA 5'end labeling, the GTP stock solution should be diluted to 1-3 times the mRNA concentration.
规格
产品名称
牛痘病毒加帽体系
分子类型
生物试剂/缓冲液
储存与运输
储存条件
室温
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