Department of Pediatrics, Ludwig-Maximilian-University of Munich, 80337 Munich, Germany.
Ethris GmbH, Planegg, 82152 Planegg, Germany.
RNA. 2019 Apr;25(4):507-518. doi: 10.1261/rna.069286.118. Epub 2019 Jan 15.
Extensive research in the past decade has brought mRNA closer to the clinical realization of its therapeutic potential. One common structural feature for all cellular messenger RNAs is a poly(A) tail, which can either be brought in cotranscriptionally via the DNA template (plasmid- or PCR-based) or added to the mRNA in a post-transcriptional enzymatic process. Plasmids containing poly(A) regions recombine in , resulting in extensive shortening of the poly(A) tail. Using a segmented poly(A) approach, we could significantly reduce recombination of plasmids in without any negative effect on mRNA half-life and protein expression. This effect was independent of the coding sequence. A segmented poly(A) tail is characterized in that it consists of at least two A-containing elements, each defined as a nucleotide sequence consisting of 40-60 adenosines, separated by a spacer element of different length. Furthermore, reducing the spacer length between the poly(A) segments resulted in higher translation efficiencies compared to homogeneous poly(A) tail and reduced recombination (depending upon the choice of spacer nucleotide). Our results demonstrate the superior potential of segmented poly(A) tails compared to the conventionally used homogeneous poly(A) tails with respect to recombination of the plasmids and the resulting mRNA performance (half-life and translational efficiency).
在过去的十年中，广泛的研究使 mRNA 更接近其治疗潜力的临床实现。所有细胞信使 RNA 的一个共同结构特征是聚（A）尾，它可以通过 DNA 模板（质粒或基于 PCR 的）共转录引入，也可以在转录后酶促过程中添加到 mRNA 中。含有聚（A）区的质粒在 中重组，导致聚（A）尾的广泛缩短。使用分段聚（A）方法，我们可以在 中显著减少质粒的重组，而对 mRNA 半衰期和蛋白表达没有任何负面影响。这种效应与编码序列无关。分段聚（A）尾的特征在于它由至少两个含 A 的元件组成，每个元件都定义为一个由 40-60 个腺苷组成的核苷酸序列，由不同长度的间隔元件隔开。此外，与均聚（A）尾相比，减少聚（A）段之间的间隔长度可提高翻译效率，并减少重组（取决于间隔核苷酸的选择）。我们的研究结果表明，与传统使用的均聚（A）尾相比，分段聚（A）尾在质粒的重组和由此产生的 mRNA 性能（半衰期和翻译效率）方面具有更好的潜力。
