Fig. 1

Engineering a TEVp responsive L7Ae. a Structure of L7Ae binding to box C/D RNA that forms a K-turn motif. TCS insertion sites in L7Ae were placed after amino acid residue N70 (L7Ae-CS1), P56 (L7Ae-CS2), or K77 (L7Ae-CS3), shown in red for each site. Protein structure was determined in³³ (PDB id: 1RLG). L7Ae-CS structure visualization was performed with pymol⁶⁵. b Schematics of L7Ae with inserted TCS translational regulation. In the absence of TEVp, L7Ae-CS binds and repress the K-turn motifs in the 5‘UTR of mRNA target (EGFP OFF-State 1). When TEVp is expressed, it cleaves the TCS, disrupting L7Ae structure and inhibiting its function (EGFP ON-State 2). c Flow cytometry analysis of three engineered L7Ae-CS tested in HEK293 cells in the absence or presence of TEVp. Data represent geometric mean of dEGFP normalized by transfection marker mKate expressed from the same constitutive promoter to account for different expression across the cell lines. ru, relative units. Significant changes determined by unpaired t-test are indicated with asterisks ***p-value < 0.001 n = 3 replicates. d Schematics of L7Ae-CS3-based HCV sensor. TEVp-scFv162¹⁶ and L7AeCS3-scFv35 fusion proteins are modules of a sensor for NS3 protein, which is associated with HCV virus. L7Ae-CS3 represses the mRNA target in the absence of NS3. In the presence NS3, binding of the intrabodies brings TEVp in proximity to L7Ae-CS3, resulting in cleavage of the RBP and derepression of the target gene. e Test of the variants of NS3 sensor in the presence and absence of BFP-nNS3¹⁶. Data represent geometric mean and standard deviation of means of dEGFP normalized by transfection marker mKate, for n = 3 replicates. Significant changes determined by unpaired t-test are indicated with asterisks *p-value < 0.05. Data collected 48 h post transfection