Simultaneous Imaging of Ribonucleic Acid and Hydrogen Sulfide in Living Systems with Distinct Fluorescence Signals Using a Single Fluorescent Probe

Abstract Ribonucleic acid (RNA) and hydrogen sulfide (H2S) are important genes and gaseous signal molecules in physiological environment. However, simultaneous investigation of distribution and interrelation of RNA and H2S in living systems is restricted by lack of functional molecular tools. To address this critical challenge, the development of TP‐MIVC is described as the first paradigm of the probes that can concurrently report ribonucleic acid and hydrogen sulfide with distinct fluorescence signals in the cancer cells, zebrafish, and living animals. The advantageous features of the probe include high stability, low background fluorescence, high sensitivity, and two‐photon imaging property. Significantly, regardless of normal mice or tumor mice, tumor tissues exhibit stronger fluorescence intensity than other organs. More interestingly, it is found that TP‐MIVC is capable of distinguishing normal mice and tumor mice by in vivo imaging. This study may open a new pathway for distinguishing malignant and benign tumor by fluorescence imaging of RNA.


Materials
Firstly, for all experiment, all reagents of synthesis and analysis experiment were obtained by commercial suppliers. Before experiment, these reagents do not further purification.
Furthermore, solvents were purified by standard methods before experiments.
Secondly, for synthesis experiment, all separation and purification of compounds were determined by TLC analysis. This method was performed on silica gel plates; In addition, column chromatography was carried out by silica gel (mesh 200-300); Silica gel was obtained from the Qingdao Ocean Chemicals.
Thirdly, for characterization of compounds, mass spectra were demonstrated by an LCQ Advantage ion trap mass spectrometer. It models is Thermo Finnigan or Agilent 1100 HPLC/MSD spectrometer; NMR spectra were obtained by the AVANCE III 400 MHz Digital NMR spectrometer.
Fourthly, for analysis experiment, ultraviolet absorption spectra were measured by a Labtech UV Power PC spectrometer; Fluorescence emission spectra were recorded with the HITACHI F4600 fluorescence spectrophotometer.
Fifthly, for biological imaging, fluorescence imaging of the cells and tissues slices was obtained with Nikon A1MP two-photon confocal microscopy. Two-photon imaging was conducted on with Nikon A1MP two-photon confocal microscopy (a Chameleon Vision II: Range 680~1080nm, a repetition rate of 80 MHz.). In vivo imaging was conducted on IVIS Lumina XR living animal imaging system.

Synthesis
Scheme. S1. The synthetic route of the probe TP-MIVC.

Preparation of Samples and Test Solution (RNA)
(1) TP-MIVC was dissolved in DMSO to get 1 mM stock solution. 10 μM TP-MIVC were used in photophysical experiments. It was obtained by adding 50 μL the stock solution to 5.0 mL buffer solution.

Preparation of Samples and Test Solution (H 2 S)
(1) TP-MIVC was dissolved in DMSO to get 1 mM stock solution. 10 μM TP-MIVC were used in photophysical experiments. It was obtained by adding 50 μL the stock solution to 5.0 mL buffer solution.

Cell viability Evaluated by MTT Assays
Firstly, HeLa cells were fed in a 96-well plate in a 5% CO 2 incubator at 37°C.
Fourthly, 96-well plate was shaken for a moment. Absorbance data at 490 nm was obtained by multiscan spectrum.
Finally, cell viability data was calculated by the following equation.