mMESSAGE mMACHINE™ 试剂盒设计用于体外合成大量的加帽 RNA。加帽 RNA 可模拟体内发现的大多数真核 mRNA，因为其在 5' 末端有一个 7-甲基鸟苷帽结构。mMESSAGE mMACHINE™ 试剂盒反应包括超高得率转录反应中的帽类似物 [m⁷G(5')ppp(5')G]。帽类似物仅作为转录本的第一个或 5' 末端 G 掺入，因为其结构使得无法在 RNA 分子中的其他位点将其掺入。

mMESSAGE mMACHINE™ 试剂盒工作原理
mMESSAGE mMACHINE™ 试剂盒有简化的反应形式，其中所有 4 种核糖核苷酸和帽类似物混合于单一溶液中。该溶液的帽类似物:GTP 比值为 4:1，这适用于尽可能提高 RNA 得率和加帽转录本比例。mMESSAGE mMACHINE™ 试剂盒适用于卵母细胞显微注射用加帽 RNA 常规合成、体外翻译、转染和其他应用。在存在高核苷酸浓度的情况下，通过优化 RNA 合成的反应条件来实现高得率。此外，合成的 RNA 不会被任何可能含有 RNase 抑制剂（酶混合物的组成部分）的污染性核糖核酸酶降解。

使用 mMESSAGE mMACHINE™ 试剂盒
在 20 µL 反应中，在 2 小时孵育期间，mMESSAGE mMACHINE™ 高得率加帽 RNA 转录将生成大量的加帽 RNA。该方法获得的得率为使用常规体外转录反应的 10–50 倍。帽类似物与 GTP 的比值已经过优化，能够在得率 (15–35 µg) 与加帽转录本的比例 (80%) 之间实现良好折衷。mMESSAGE mMACHINE™ 试剂盒还包含一份 LiCl 沉淀溶液，该溶液可高效分离来自加帽 RNA 的蛋白和未掺入的核苷酸（包括游离帽类似物），提高翻译效率。mMESSAGE mMACHINE™ 试剂盒仅针对与试剂盒中包含的聚合酶配合使用进行了优化。使用不同的聚合酶可能会导致得率低。mMESSAGE mMACHINE™ 试剂盒包含 25 次转录反应所需的所有缓冲液和试剂。使用试剂盒提供的对照模板（非洲蟾蜍延伸因子 1α，pTRI Xef），每次 mMESSAGE mMACHINE™ 反应将使用 T3 或 T7 RNA 聚合酶生成约 20–30 µg 的 RNA，或使用 SP6 RNA 聚合酶生成约 15–25 µg RNA。 

 

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mMESSAGE mMACHINE kits are designed for the in vitro synthesis of large amounts of capped RNA. Capped RNA mimics most eukaryotic mRNAs found in vivo, because it has a 7-methyl guanosine cap structure at the 5' end. mMESSAGE mMACHINE kit reactions include cap analog [m7G(5')ppp(5')G] in an ultra high-yield transcription reaction. The cap analog is incorporated only as the first or 5' terminal G of the transcript because its structure precludes its incorporation at any other position in the RNA molecule.

How mMESSAGE mMACHINE kits work

mMESSAGE mMACHINE kits have a simplified reaction format in which all four ribonucleotides and cap analog are mixed in a single solution. The cap analog:GTP ratio of this solution is 4:1, which is optimal for maximizing both RNA yield and the proportion of capped transcripts. mMESSAGE mMACHINE kits are ideal for the routine synthesis of capped RNAs for oocyte microinjection, in vitro translation, transfection, and other applications. The high yields are achieved by optimizing reaction conditions for RNA synthesis in the presence of high nucleotide concentrations. In addition, the RNA synthesized is protected from degradation by any contaminating ribonucleases that may be present with RNase inhibitor—a component of the Enzyme Mix.

Using mMESSAGE mMACHINE Kits

In a 20 μL reaction during a two hour incubation, mMESSAGE mMACHINE high yield capped RNA transcription will yield large mass amounts of capped RNA. Up to 10–50 times the yield obtained with conventional in vitro transcription reactions. The ratio of cap analog to GTP has been optimized to allow the best compromise between yield (15–35 μg) and proportion of transcripts that are capped (80%).

mMESSAGE mMACHINE kits also contain a LiCl precipitation solution that is efficient for separating proteins and unincorporated nucleotides (including free cap analog) from the capped RNA, allowing an increased efficiency of translation. mMESSAGE mMACHINE kits are only optimized for use with the polymerases included in the kit. Using a different polymerase may result in low yields. The mMESSAGE mMACHINE kits contain all the buffers and reagents necessary for 25 transcription reactions. Using the control template supplied with the kits (Xenopus elongation factor 1α, pTRI Xef), each mMESSAGE mMACHINE reaction will yield approximately 20–30 μg of RNA using T3 or T7 RNA polymerase or about 15–25 μg RNA using SP6 RNA polymerase.

 

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