Fig. 1: Principle and schematic workflow for the sequencing-based identification of functional riboswitches in human cells. | Nature Communications

Fig. 1: Principle and schematic workflow for the sequencing-based identification of functional riboswitches in human cells.

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

Fig. 1

a HEK-293 cells are transfected with a plasmid library harboring PCR-binding site (red arrowheads)-flanked aptazyme variants with a randomized motif (here: tetracycline (Tet)-hammerhead design). b Functional ON-switches are characterized by aptazyme auto-cleavage and mRNA-degradation in the absence of Tet, whereas upon Tet addition, cleavage is inhibited, resulting in stable mRNA. c Following cell lysis, RNA purification and reverse transcription into cDNA, riboswitch sequences are PCR-amplified and applied to amplicon-seq analysis. Functional sequences are identified from differential expression analyses (stimulated vs. unstimulated). ITR, inverted terminal repeat; CMV, cytomegalovirus; GFP, green fluorescent protein; pA, polyadenylation signal.

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