All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR or DNA cloning. The oligonucleotide design, however, is a challenge to non-experts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the Rational design of Oligonucleotides, Calculated with Kinetic parameters for Enhanced in vitro Transcription (ROCKET). The Python tool employs thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.

中文翻译：

---

合理设计寡核苷酸以增强小RNA的体外转录

各种RNA分子可以通过固相化学合成、引物延伸、PCR或DNA克隆获得的DNA模板，使用T7 RNA聚合酶进行体外转录产生。然而，寡核苷酸设计对于非专家来说是一个挑战，因为这依赖于随着时间的推移凭经验建立的一组规则。在这里，我们描述了一个 Python 程序，以促进寡核苷酸的合理设计，并使用增强体外转录 (ROCKET) 的动力学参数进行计算。Python 工具采用热力学参数，执行折叠能量计算，并选择适合聚合酶延伸反应的寡核苷酸。这些寡核苷酸提高了模板 DNA 的产量。利用程序选择的寡核苷酸，可以通过DNA聚合酶延伸反应和转录反应的一锅反应来制备tRNA转录本。此外，由于模板 DNA 合成的增强，ROCKET 选择的寡核苷酸比 Primerize（一种用于设计体外转录寡核苷酸的领先软件）选择的寡核苷酸提供更高的转录产量。除了测试的 50 多个 tRNA 基因外，还发现一种体外转录的自切割核酶具有催化活性。此外，该程序还可应用于mRNA的合成，展示了ROCKET软件的广泛适用性。