Benjamin Strobel, Maike Spöring, Holger Klein, Dragica Blazevic, Werner Rust et al.
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 × 10^4 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, this method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches.
Benjamin Strobel, Maike Spöring, Holger Klein, Dragica Blazevic, Werner Rust, Sergi Sayols, Jörg S. Hartig, Sebastian Kreuz. High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells. 2020-02-05. https://doi.org/10.1038/s41467-020-14491-x
A pooled guanine-responsive library tested 4,096 hepatitis-delta-virus ribozyme constructs and 16,384 hammerhead ribozyme constructs in the CMV-eGFP 3′ UTR of transiently transfected HEK-293H cells. Self-barcoding cDNA amplicon counts were measured after 3 h untreated, 30 µM, 100 µM, or 300 µM guanine stimulation, with eight biological replicates per condition.
A 4,096-member synthetic library randomized six nucleotides in the P1/U1-binding communication region of a guanine-responsive aptamer-RNA device and was screened in HEK-293H cells. The processed table preserves the published source-data statistics for untreated, 30 µM, 100 µM, and 300 µM guanine stimulation, with eight biological replicates per condition.
An episomal CMV-eGFP 3′-UTR library containing all 16,384 combinations of a seven-nucleotide communication module was screened in HEK-293H cells. Self-barcoding cDNA amplicon counts were measured after 3 h untreated, 25 µM, or 50 µM tetracycline stimulation, with eight biological replicates per condition.
Nine synthetic Twister sublibraries (2N3N, 3N2N, 3N3N and CG/noCG variants; 18,432 constructs total) were screened in HEK-293H cells. Self-barcoding cDNA amplicon counts were measured after 3 h untreated, 12.5 µM, 25 µM, or 50 µM tetracycline stimulation, with eight biological replicates per condition.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
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