Kobori Shungo, Nomura Yoko, Miu Anh, Yokobayashi Yohei
Department of Biomedical Engineering, University of California, Davis, CA 95616, USA.
Department of Biomedical Engineering, University of California, Davis, CA 95616, USA Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904 0495, Japan
Nucleic Acids Res. 2015 Jul 27;43(13):e85. doi: 10.1093/nar/gkv265. Epub 2015 Mar 30.
Self-cleaving ribozymes are found in all domains of life and are believed to play important roles in biology. Additionally, self-cleaving ribozymes have been the subject of extensive engineering efforts for applications in synthetic biology. These studies often involve laborious assays of multiple individual variants that are either designed rationally or discovered through selection or screening. However, these assays provide only a limited view of the large sequence space relevant to the ribozyme function. Here, we report a strategy that allows quantitative characterization of greater than 1000 ribozyme variants in a single experiment. We generated a library of predefined ribozyme variants that were converted to DNA and analyzed by high-throughput sequencing. By counting the number of cleaved and uncleaved reads of every variant in the library, we obtained a complete activity profile of the ribozyme pool which was used to both analyze and engineer allosteric ribozymes.
自我切割核酶存在于生命的所有领域，被认为在生物学中发挥着重要作用。此外，自我切割核酶一直是合成生物学应用中广泛工程改造的对象。这些研究通常涉及对多个通过合理设计或通过筛选发现的单个变体进行费力的检测。然而，这些检测仅提供了与核酶功能相关的大片段序列空间的有限视角。在此，我们报告了一种策略，该策略能够在单个实验中对1000多个核酶变体进行定量表征。我们生成了一个预定义核酶变体库，将其转化为DNA并通过高通量测序进行分析。通过计算文库中每个变体的切割和未切割读数的数量，我们获得了核酶库的完整活性图谱，该图谱用于分析和工程化变构核酶。
