Fig. 3: Fluorophores with trifluoroethyl substituent at N1 position markedly enhance Squash fluorescence in vitro and in vivo.
From: Near-infrared fluorogenic RNA for in vivo imaging and sensing
a Schematic drawing of Squash-binding fluorophores and their derivatives with trifluoroethyl substitutions at the N1 position. To enhance the brightness and red-shift the fluorescence spectra of Squash-binding fluorophores (left), we synthesized these fluorophore derivatives with trifluoroethyl at the N1 position (RFP-mimics-1T, right), including DFHBI-1T, DFHO-1T, DFIM-1T, DFAME-1T, DFQL-1T. b Excitation and emission spectra of Squash bound to RFP-mimics-1T. Excitation (Ex, dotted line) and emission (Em, solid line) spectra were measured using in vitro transcribed Squash RNA (10 μM) and RFP-mimics-1T (1 μM) in buffer. In particular, Squash:DFQL-1T complex exhibits NIR fluorescence (Ex=516 nm, Em= 654 nm). c RFP-mimics-1T exhibit improved fluorescence compared to RFP-mimics when binding to Squash. We incubated Squash (1 μM) with different Squash-binding fluorophores (RFP-mimics or RFP-mimics-1T, 10 μM) and measured the fluorescence. The RFP-mimics-1T exhibited increased fluorescence compared to the original RFP-mimics. Data represent mean values  ±  s.d. for n  =  3 independent experiments. d RFP-mimics-1T exhibit increased Squash fluorescence in living cells. HEK293T cells expressing circular Squash RNA were imaged in the presence of 2 μM RFP-mimics or RFP-mimics-1T. RFP-mimics-1T exhibit increased Squash fluorescence in living cells compared to RFP-mimics. Exposure time: 500 ms for RFP-mimics or RFP-mimics-1T and 100 ms for Hoechst. Imaging filters: DFHBI-1T/DFHBI (Ex = 488 nm, Em = 525 ± 25 nm), DFHO-1T/DFHO/DFIM-1T/DFIM (Ex = 514 nm, Em = 575 ± 20 nm), DFAME-1T/DFAME (Ex = 561 nm, Em = 617 ± 32.5 nm), DFQL-1T/DFQL (Ex = 514 nm, Em = 685 ± 20 nm). Scale bar, 20 µm. e Quantification of average brightness of Squash-expressing cells incubated with different fluorophores. Background fluorescence was measured in control cells with fluorophores alone (10 μM). Data represent mean values  ±  s.d. for n = 100 cells per condition. Statistical significances were determined by a two-tailed Student’s t test. ****p-values: 4.8 × 10−36, 3.9 × 10−82, 1.8 × 10−71, 4.1 × 10−95, 1.0 × 10−95, in left-to-right order. f, g Imaging of transplanted mice using Squash:DFQL-1T system. The results showed that mice incubated with Squash-expressing cells exhibited obvious NIR fluorescence signal (top row), while mice incubated with control cells exhibited minimal fluorescence in the presence of DFQL-1T. Image acquisition time, 500 ms. Data represent mean values  ±  s.d. for n  =  3 independent experiments.
