RNA molecules can be conveniently synthesized in vitro by the T7 RNA polymerase (T7 RNAP). In some experiments, such as co-transcriptional biochemical analyses, continuous synthesis of RNA is not desired. Here, we propose a method for a single-pass transcription that yields a single transcript per template DNA molecule using the T7 RNAP system. We hypothesized that stalling the polymerase downstream of the promoter region and subsequent cleavage of the promoter by a restriction enzyme (to prevent promoter binding by another polymerase) would allow synchronized production of a single transcript per template. The single-pass transcription was verified in two different scenarios: a short self-cleaving ribozyme and a long mRNA. The results show that a controlled single-pass transcription using T7 RNAP allows precise measurement of co-transcriptional ribozyme activity, and this approach will facilitate the study of other kinetic events.

中文翻译：

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通过 T7 RNA 聚合酶进行单次转录

RNA 分子可以通过 T7 RNA 聚合酶 (T7 RNAP) 在体外方便地合成。在一些实验中，例如共转录生化分析，不需要连续合成 RNA。在这里，我们提出了一种单程转录方法，该方法使用 T7 RNAP 系统为每个模板 DNA 分子生成一个转录本。我们假设在启动子区域下游停止聚合酶并随后通过限制酶切割启动子（以防止启动子被另一种聚合酶结合）将允许每个模板同步产生单个转录本。单程转录在两种不同的情况下得到验证：短的自切割核酶和长的 mRNA。