Research Beyond Borders, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str. 65, 88397, Biberach an der Riss, Germany.
Department of Chemistry, University of Konstanz, Universitätsstraße 10, 78464, Konstanz, Germany.
Nat Commun. 2020 Feb 5;11(1):714. doi: 10.1038/s41467-020-14491-x.
Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including next-generation gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires large-scale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8 × 10 constructs) by cDNA-amplicon-sequencing in transiently transfected and stimulated human cells. The self-barcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches.
介导配体依赖性 RNA 切割或剪接调节的合成核糖开关代表了控制基因表达的精巧工具，在包括下一代基因治疗在内的各种应用中具有广泛的应用前景。然而，由于对上下文相关的结构-功能关系的理解有限，功能性核糖开关的鉴定需要对适体-效应结构域设计进行大规模筛选，而这受到缺乏合适的细胞高通量方法的限制。在这里，我们描述了一种快速且广泛适用的方法，通过瞬时转染和刺激的人类细胞中的 cDNA 扩增子测序，对复杂的核糖开关文库（~1.8×10 个构建体）进行功能筛选。每个构建体的自我编码性质允许在没有额外的预选择或 cDNA 操作步骤的情况下对差异 mRNA 水平进行定量。我们应用这种方法来设计基于锤头状核酶、丙型肝炎病毒和 Twister 核酶以及 U1-snRNP 多聚腺苷酸化依赖性 RNA 器件的四环素和鸟嘌呤响应的 ON 和 OFF 开关。总之，我们的方法能够快速有效地进行高通量核糖开关鉴定，从而克服了治疗应用基因开关开发级联中的一个主要障碍。
