pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences.
pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences.
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DOI：
10.1038/mtna.2016.21
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发表时间：
2016-04-19
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对体外转录（IVT）mRNA的大规模合成的日益增长的需求是由mRNA在细胞工程和治疗应用中用于瞬时基因表达的日益增长的使用所驱动的。IVT mRNA效力的一个重要决定因素是3′聚腺苷（poly（A））尾，其长度与翻译效率相关。然而，目前用于产生IVT mRNA的方法依赖于源自环状质粒或PCR产物的模板，其中均聚片段是不稳定的，因此将编码的聚腺苷酸尾长度限制为约120个碱基对（bp）。在这里，我们已经开发了一种新的方法，使用先前描述的线性质粒系统，pJazz的扩展聚（A）tracts的生成。我们发现，线性质粒可以成功地繁殖聚（A）束长度约500 bp的IVT mRNA的生产。然后，我们通过去除外来限制性位点，在延伸的多克隆位点上游添加T7启动子序列，并在编码的poly（A）区下游添加独特的IIS型限制性位点来修饰pJazz，以促进具有精确定义的编码poly（A）区和3′末端的IVT mRNA的产生。所得质粒（命名为pEVL）可用于产生具有一致的限定长度和末端残基的IVT mRNA。
Increasing demand for large-scale synthesis of in vitro transcribed (IVT) mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3′ polyadenosine (poly(A)) tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A) tail lengths to ~120 base pairs (bp). Here, we have developed a novel method for generation of extended poly(A) tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A) tracts up to ~500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A) tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A) tracts and 3′ termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s).