A universal method to produce in vitro transcripts with homogeneous 3′ ends -: art. no. e56
A universal method to produce in vitro transcripts with homogeneous 3′ ends -: art. no. e56
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DOI：
10.1093/nar/gnf055
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发表时间：
2002-06-15
影响因子：
14.9
通讯作者：
Mörl, M
中科院分区：
文献类型：
--
作者：
Schürer, H;Lang, K;Mörl, M
本文描述了一种方法，该方法可以克服体外转录测定的一般缺点：RNA聚合酶倾向于在RNA 3'端添加额外的核苷酸，这些核苷酸在线性化的DNA模板中没有编码。此外，这些聚合酶在靠近RNA的3'端处显示出相当高的过早终止率。这些特征导致全长转录物的产量下降，往往使确定和分离正确转录的全长RNA变得困难。锤头核酶在cis中经常被用来切割这些额外的核苷酸。然而，这种核酶的上游序列要求限制了它的一般可用性。相反，丁型肝炎病毒核酶没有这样的要求，因此可以顺式应用于任何RNA序列。由于核酶的催化活性，所需的转录物被释放为具有均匀3'端的RNA分子。释放的RNA的2',3'-环磷酸基团可以通过T4多核苷酸激酶处理轻松有效地去除。所提出的方法可以应用于几乎任何要转录的序列，因此优于其他核酶策略，这表明在需要具有均匀文德的转录本的每个领域都有可能应用。
A method is described that allows a general drawback of In vitro transcription assays to be overcome: RNA polymerases tend to add extra nucleotides to the RNA 3' end that are not encoded In the linearized DNA template. Furthermore, these polymerases show a considerable rate of premature termination close to the RNA's 3' end. These features lead to a decreased yield of full-length transcripts and often make it difficult to determine and Isolate the correctly transcribed full-length RNA. The hammerhead ribozyme Is frequently used In cis to cleave off these extra nucleotides. However, the upstream sequence requirements of this ribozyme restrict Its general usability. In contrast, the hepatitis delta virus ribozyme has no such requirements and can therefore be applied to any RNA sequence In cis. Due to the catalytic activity of the ribozyme, the desired transcript is released as an RNA molecule with a homogeneous 3' end. The resulting 2',3'-cyclophosphate group of the released RNA can be easily and efficiently removed by T4 polynucleotide kinase treatment. The presented method can be applied for virtually any sequence to be transcribed and is therefore superior to other ribozyme strategies, suggesting possible applications In every field where transcripts with homogeneous Wends are required.