Fig. 1

Visualizing RNAs live with Riboglow-FLIM-Cy5. (A) Cobalamin (Cbl) is synthetically linked to Cy5, quenches its fluorescence, binds an RNA ligand (called the “A-tag” throughout), and alters its fluorescence lifetime. The RNA tag is genetically fused to an RNA of interest. Fluorescence lifetime decay data are fit by multiexponential reconvolution for a defined region of interest (ROI), yielding component and average lifetime (both are amplitude-weighted lifetimes, see methods for details). Both can be illustrated by a false-color scale. The "component lifetime" (v) resolves the lifetime pixel-by-pixel, while the "average lifetime" (vi, vii) provides one lifetime value per ROI. (B) Riboglow-FLIM-Cy5 in live cells (4 independent experiments, 44 cells, 1 symbol = 1 cell). One-way ANOVA (95% confidence limit); post hoc test (Tukey HSD), p-value listed (∗∗∗∗p ≤ 0.0001). Error bars: mean and standard deviation (±SD). (C) Representative cells from (B), component lifetimes displayed. Scale bars, 10 μm. (D) Phasor plot showing graphical representation of lifetime coordinates G and S for all data shown in (B). Light blue circle, untransfected cells; pink circle, transfected cells producing 1/8-NORAD(4A), 4 independent experiments, 44 cells. Phasor representative images shown in Fig. S2 D.