Cis-acting ribozymes for the production of RNA in vitro transcripts with defined 5' and 3' ends.
Cis-acting ribozymes for the production of RNA in vitro transcripts with defined 5' and 3' ends.
复制标题
DOI：
10.1007/978-1-62703-113-4_7
复制
发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Walker, Scott C
中科院分区：
文献类型：
--
作者：
Avis, Johanna M;Conn, Graeme L;Walker, Scott C
体外转录RNA的使用通常受到5'端序列限制以及5'端和3'端可能发生的转录异质性问题的限制。本章介绍了顺式作用核酶、5'端锤头状病毒 (HH) 和 3' 端丁型肝炎病毒 (HDV) 的使用，用于直接转录处理以产生具有精确定义末端的靶 RNA。该方法的重点是使用 pRZ 和 p2RZ 质粒，这些质粒旨在简化此类双核酶模板的生产。这些质粒各自带有一个经过独特限制性位点修饰的 3'-HDV，该限制性位点允许核酶保留在质粒上，因此可以从克隆过程中省略。详细介绍了设计针对每个目标 RNA 5' 端的独特锤头核酶所需的额外步骤。在大多数情况下，可以通过将单个 PCR 产物克隆到 pRZ 或 p2RZ 载体中来获得带有 5'-HH 核酶和 3'-HDV 核酶的转录模板。还描述了优化这些模板的转录产量和分离均质目标 RNA 的方案。
The use of in vitro transcribed RNA is often limited by sequence constraints at the 5'-end and the problem of transcript heterogeneity which can occur at both the 5'- and 3'-ends. This chapter describes the use of cis-acting ribozymes, 5'-end hammerhead (HH) and 3'-end hepatitis delta virus (HDV), for direct transcriptional processing to yield target RNAs with precisely defined ends. The method is focused on the use of the pRZ and p2RZ plasmids that are designed to simplify the production of such dual ribozyme templates. These plasmids each bear a 3'-HDV modified with a unique restriction site that allows the ribozyme to remain on the plasmid and, therefore, be omitted from the cloning procedure. The additional steps required to design a unique hammerhead ribozyme tailored to the 5'-end of each target RNA are detailed. In most cases, a transcriptional template bearing a 5'-HH ribozyme and a 3'-HDV ribozyme can be achieved by cloning a single PCR product into either the pRZ or p2RZ vector. Protocols for optimization of transcription yields from these templates and the isolation of the homogeneous target RNA are also described.