General plasmids for producing RNA in vitro transcripts with homogeneous ends -: art. no. e82
General plasmids for producing RNA in vitro transcripts with homogeneous ends -: art. no. e82
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DOI：
10.1093/nar/gng082
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发表时间：
2003-08-01
影响因子：
14.9
通讯作者：
Conn, GL
中科院分区：
文献类型：
--
作者：
Walker, SC;Avis, JM;Conn, GL
噬菌体RNA聚合酶（RNAPs），如T7 RNAP的体外转录产物，常常在3'端存在相当程度的异质性，并且对于某些启动子序列，在5'端也存在异质性。对于某些应用而言，这种转录产物的异质性造成了严重的问题。一种可能的解决方案是在目标RNA序列的5'端和/或3'端将核酶整合到转录产物中。这种方法已被广泛应用，但对于每一种要研究的新RNA，都需要构建新的转录载体或由PCR衍生的模板。为了克服这一局限，我们构建了两种用于产生均一RNA转录产物的通用质粒：一种编码3' - 丁型肝炎病毒（HDV）核酶，另一种与两步PCR结合使用，能够产生双[5' - 锤头（HH）和3' - HDV]核酶构建体。一系列可供选择的克隆和转录终止线性化限制酶位点确保了几乎任何RNA序列都能够从这些质粒中被克隆和转录。对于所有测试的RNA序列，都获得了较高产量的转录产物。这些质粒为简单、快速构建新的编码RNA的质粒提供了工具，以便为所有应用生产毫克量的均一的体外转录产物。
In vitro transcripts of bacteriophage RNA polymerases (RNAPs), such as T7 RNAP, often suffer from a considerable degree of 3'-end heterogeneity and, with certain promoter sequences, 5'-end heterogeneity. For some applications, this transcript heterogeneity poses a significant problem. A potential solution is to incorporate ribozymes into the transcripts at the 5'- and/or 3'-end of the target RNA sequence. This approach has been used quite widely but has required the generation of new transcription vectors or PCR-derived templates for each new RNA to be studied. To overcome this limitation, we have created two general plasmids for producing homogeneous RNA transcripts: one encodes a 3'- hepatitis delta virus (HDV) ribozyme and the other, used in combination with a two-step PCR, allows the production of double [5'-hammerhead (HH) and 3'-HDV] ribozyme constructs. A choice of cloning and run-off transcription linearisation restriction enzyme sites ensures that virtually any RNA sequence can be cloned and transcribed from these plasmids. For all the RNA sequences tested, good yields of transcript were obtained. These plasmids provide the tools for the simple, rapid creation of new RNA-coding plasmids to produce milligram quantities of homogeneous in vitro transcripts for all applications.