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High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

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STROBEL, Benjamin, Maike SPÖRING, Holger KLEIN, Dragica BLAZEVIC, Werner RUST, Sergi SAYOLS, Jörg S. HARTIG, Sebastian KREUZ, 2020. High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells. In: Nature Communications. Nature Publishing Group. 11(1), 714. eISSN 2041-1723. Available under: doi: 10.1038/s41467-020-14491-x

@article{Strobel2020-02-05Hight-49314, title={High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells}, year={2020}, doi={10.1038/s41467-020-14491-x}, number={1}, volume={11}, journal={Nature Communications}, author={Strobel, Benjamin and Spöring, Maike and Klein, Holger and Blazevic, Dragica and Rust, Werner and Sayols, Sergi and Hartig, Jörg S. and Kreuz, Sebastian}, note={Article Number: 714} }

Strobel, Benjamin 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<sup>4</sup> 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. High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells Kreuz, Sebastian Klein, Holger Blazevic, Dragica Sayols, Sergi Kreuz, Sebastian 2020-04-28T08:25:28Z Rust, Werner Blazevic, Dragica Klein, Holger Spöring, Maike Strobel, Benjamin Hartig, Jörg S. eng Rust, Werner Hartig, Jörg S. 2020-02-05 Spöring, Maike 2020-04-28T08:25:28Z Sayols, Sergi

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