Fig. 5: Generation of Squash-based NIR fluorescent sensor for ncRNAs in vivo.
From: Near-infrared fluorogenic RNA for in vivo imaging and sensing
a Design of the Squash-based ncRNA sensors. The ncRNAs probe (green), stem ___domain, and Squash ___domain (beige) are depicted. The stem ___domain is composed of Squash aptamer and target sequence that form a short helix. Target ncRNA binding to the antisense probe would form a longer and stabler duplex than the original stem, thus inducing the folding of Squash aptamers and NIR fluorescence of DFQL-1T. b, c Optimization of the stems for the ncRNA sensors. We generated the microRNA-21 and piRNA-36026 sensors with different stems by in vitro transcription, respectively. Sensors with different stems (5 µM) were incubated with DFQL-1T (10 µM) in the presence or absence of targets (10 µM). The optimal microRNA-21 sensor containing stem 3 and piRNA-36026 sensor containing stem 4 showed a 6-fold and 5-fold increase in fluorescence upon target incubation, respectively. The optimal stem is indicated in the black box. Data represent mean values  ±  s.d. for n  =  3 independent experiments. d, e Imaging endogenous ncRNAs with Squash-based ncRNA sensors. To determine if the Squash-based ncRNA sensors can image ncRNAs in living cells, we expressed Squash in LO2 and MCF-7. Expression levels of miRNA-21 and piRNA 36026 in MCF-7 are significantly higher compared to LO2. Cell culture media was then replaced with phenol red-free DMEM containing DFQL-1T (10 μM) for 1 h before imaging. NIR fluorescence can be readily observed in MCF-7 cells, and the corresponding ncRNA inhibitors can reduce the fluorescence to a minimum level. Image acquisition time, 500 ms. Scale bar, 20 µm. Data represent mean values  ±  s.d. for n  =  3 independent experiments. f, g Squash-based NIR fluorescent sensor for ncRNAs imaging in vivo. Cells expressing ncRNA sensors (108 cells) were transplanted into mice, and then injected with DFQL-1T (10 µM) in situ for imaging. Sensor-expressing MCF-7 cells exhibit significantly higher NIR fluorescence (bottom row) compared to LO2 cells (top row). Image acquisition time, 500 ms. Data represent mean values  ±  s.d. for n  =  3 independent experiments.
