Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA
Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA
复制标题
DOI：
10.1021/jacs.7b03981
复制
发表时间：
2017-07-26
影响因子：
15
通讯作者：
Romesberg, Floyd E.
中科院分区：
文献类型：
--
作者：
Chen, Tingjian;Romesberg, Floyd E.
对RNA和修饰的RNA寡核苷酸的需求不断增加，但与DNA寡核苷酸相比，它们通常化学合成昂贵得令人望而却步，并且与较长的RNA不同，它们仅通过体外转录低效地产生，特别是当修饰时。为了解决这些挑战，我们以前报道了一种热稳定的DNA聚合酶SFM 4 -3的进化，它能更有效地接受具有2 '-取代基的底物。我们现在表明，SFM 4 -3有效地转录RNA或2 '-F-修饰的RNA，并且它还有效地PCR扩增混合RNA和DNA组合物的寡核苷酸。此外，与热循环和使用一种新的DNA模板，我们证明了聚合酶链式转录（PCT）反应，导致在数量级更多的RNA或修饰的RNA的指数生产比传统的转录。PCT比常规转录更有效和通用，并且可以以化学合成成本的一小部分产生大量的任何RNA或修饰的RNA寡核苷酸。
There is increasing demand for RNA and modified RNA oligonucleotides, but in contrast to DNA oligonucleotides, they are typically prohibitively expensive to chemically synthesize, and unlike longer RNAs, they are only inefficiently produced by in vitro transcription, especially when modified. To address these challenges, we previously reported the evolution of a thermostable DNA polymerase, SFM4-3, that more efficiently accepts substrates with 2'-substituents. We now show that SFM4-3 efficiently transcribes RNA or 2'-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. In addition, with thermocycling and the use of a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription. PCT is more efficient and general than conventional transcription and can produce large amounts of any RNA or modified RNA oligonucleotide at a fraction of the cost of chemical synthesis.