Fig. 2: Preparation of highly fluorogenic TMP-Tag ligands.
a Chemical structures of TMP-Tag ligands consisting of Trimethoprim tethered rhodamines with varying linker length. b Absorption and emission spectra of MaP618m-TMP (250 nM) in the presence (plain line) and absence (dashed line) of TMP-Tag (500 nM) after 1 h incubation. c Fluorescent response of TMP-Tag/Halo-Tag ligands (250 nM, gray dots) upon binding to TMP-Tag/Halo-Tag (500 nM, red boxes). d Binding modes of TMP ligands with TMP-Tag (PDB ID: 7NAE). e Wash-free live-cell confocal images of co-cultured untransfected U2OS cells and U2OS SNAP-TMP-NLS-expressing cells labeled with fluorescent ligands (200 nM or 10 μM) for 30 min. Untransfected U2OS cells are marked with white dashed lines. The calibration bar was indicated in the upper right corner. f Statistical charts of signal-over-background signals (S/B) and nuclear brightness of live U2OS imaged in (e). ** indicates statistical significance (p = 0.0015, p < 0.01), not significant (n.s.), significant: p ≤ 0.0001 (****), n = 20 cells (S/B) and 15 cells (brightness) from three individual experiments. Chemical structures of TMR (g) and SiR (l) derivatives coupled to TMP-Tag substrate. Absorption and emission spectra of TMR (h) and SiR (m) -derived TMP probes (1 μM) in the presence (plain line) and absence (dashed line) of TMP-Tag (2 μM) after 1 h incubation. i, n Wash-free live-cell confocal images of co-cultured untransfected U2OS cells and U2OS SNAP-TMP-NLS-expressing cells labeled with TMR-TMP, SiR-TMP, MaP555-TMP, and MaP655-TMP for 30 min. j, o Statistical charts of signal-over-background signals (S/B) and nuclear brightness of live U2OS imaged in (i, n). The cells were pre-labeled with 350 nM SiR-BG (j) or 200 nM R500-BG (o) for 2 h prior to the treatment with 200 nM TMP probes. Normalized fluorescence intensities of TMR-derived (k) or SiR-derived (p) ligands at different time points for nuclear protein labeling in live U2OS cells. In e, i, n, the untransfected cells are marked with white dashed lines. The white numbers indicate the average fluorescence intensity ratio between the nuclear signal (transfected cells) and the cytosol signal (normal cells) (Fnuc/Fcyt). The yellow numbers indicate the average fluorescence intensity ratio between the cytosol signal (normal cells) and the buffer signal (Fcyt/Fbuf). n = 20 cells from three individual experiments, mean values ± S.E.M. Significance was calculated using two-sided t-tests, not significant (n.s.), significant: p ≤ 0.0001 (****). In e, i, n, Scale bar: 10 μm. Box-and-whisker plots in (j, o): Center line represents the median; whiskers extend from minimum to maximum values; all individual data points are shown. Source data are provided as a Source Data file.
