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使用反式作用基因组δ核酶生成具有均一3'末端的体外转录本。

Generating in vitro transcripts with homogenous 3' ends using trans-acting antigenomic delta ribozyme.

作者信息

Wichlacz Agnieszka, Legiewicz Michał, Ciesiołka Jerzy

机构信息

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.

出版信息

Nucleic Acids Res. 2004 Feb 18;32(3):e39. doi: 10.1093/nar/gnh037.

DOI:10.1093/nar/gnh037
PMID:14973333
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC373431/
Abstract

In most in vitro run-off transcription reactions with T7 RNA polymerase, transcripts with heterogeneous ends are commonly obtained. Towards the goal of finding a simple and effective procedure for correct processing of their 3' ends we propose the use of trans-acting antigenomic delta ribozyme. We demonstrate that the extension of nascent transcripts with only seven nucleotides complementary to the ribozyme's recognition site, and subsequently, the removal of those nucleotides with the ribozyme acting in trans, is an efficient procedure for generating transcripts with homogenous 3' ends. This approach was tested on two model RNA molecules: an in vitro transcript of yeast tRNA(Phe) and a delta ribozyme, which processed itself during transcription. The proposed procedure is a simple alternative to the use of ribozymes as cis-cleaving autocatalytic cassettes attached to transcript 3' ends. As there is little possibility that the required additional stretch, only seven nucleotides long, enters into stable interactions with other parts of the transcripts, it can be cleaved off with high efficacy.

摘要

在大多数使用T7 RNA聚合酶的体外连续转录反应中，通常会获得末端不均一的转录本。为了找到一种简单有效的方法来正确处理其3'末端，我们建议使用反式作用的反基因组δ核酶。我们证明，新生转录本仅延伸七个与核酶识别位点互补的核苷酸，随后用反式作用的核酶去除这些核苷酸，是产生具有均一3'末端转录本的有效方法。该方法在两个模型RNA分子上进行了测试：酵母tRNA(Phe)的体外转录本和一个在转录过程中自我加工的δ核酶。所提出的方法是使用核酶作为连接到转录本3'末端的顺式切割自催化盒的简单替代方法。由于所需的仅七个核苷酸长的额外延伸与转录本的其他部分形成稳定相互作用的可能性很小，因此它可以被高效切割掉。