Extended Data Fig. 1: Screening, optimization and characterization of Clivia.
(a) Excitation and emission spectra of NBSI in glycerol. NBSI showed minimal fluorescence in water, but emitted bright fluorescence in glycerol. Δλ represents the Stokes shift. (b) The Mfold-predicted secondary structure of R8 aptamer. (c) Fluorescence activation of NBSI by R8 aptamer. (d) The excitation and emission spectra of R8-NBSI complex and NBSI alone. (e) FACS analysis of R8-NBSI complexes in BL21 Star™ (DE3) cells. E. coli. BL21 Star™ (DE3) cells expressing tRNA-R8 were induced by adding IPTG at 37 °C for 4 h. The cells were collected and incubated with 0.5 µM NBSI in HEPES buffer (containing 5 mM Mg2+). Fluorescence was analyzed using a flow cytometry with a 561 nm excitation. The gate (blue) was placed based on the BL21 Star™ (DE3) cells transformed with empty vector and incubated with 0.5 µM NBSI to determine population fraction that expressed tRNA-R8. (f) The Mfold-predicted secondary structures of R8 and its truncation mutants. (g) Quantification of NBSI fluorescence induced by R8 and its truncation mutants. The markedly reduced fluorescence of the T5 aptamer suggests that T4 is the minimal aptamer sequence required for activation of NBSI fluorescence. (h) Stem-loop indicated in R8-T4 structure was replaced by other stem-loops with difference sequences. (i) Quantification of fluorescence of R8-T4 with different stem-loops. (j) Mutation sites (purple nucleotides) in T4-2. (k) Quantification of fluorescence of T4-2 mutants. (l) Thermostability of different T4-2 mutants. (m) Potassium independence for Clivia-induced fluorescence. (n) Schematic representation of different tandem arrays of Clivia. (o) Live cell imaging of different tandem arrays of Clivia in HEK293T cells. The cells were incubated with 0.2 µM NBSI for 10 min before imaging. Scale bar, 10 μm. (p) Quantification analysis of the fluorescence for different Clivia arrays in live cells. Data represent the mean ± s.d. (n = 200 cells). Data in (c), (g), (i), (k), (l), (m) represent the mean ± s.d. from three biologically independent replicates.
