Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc03150e Click here for additional data file.

Investigating the many roles RNA plays in cellular regulation and function has increased demand for tools to explore RNA tracking and localization within cells.


General Methods
All reagents used for the synthesis of PreQ1-TO derivatives were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. All DNA and RNA Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). All restriction enzymes, bio-reagents, nucleotide stains, and competent bacterial strains were purchased from New England Biolabs (Ipswitch, MA), Promega (Madison, WI), or Life Technologies (Carlsbad,CA). 1 H and 13 C NMR spectra were recorded on a Varian VX 500 MHz NMR Spectrometer. High resolution mass spectroscopy was collected on an Agilent Infinity 1260 LC and tandem Agilent 6230 high resolution time of flight (TOF) mass spectrometer managed by the UCSD Department of Chemistry and Biochemistry Molecular Mass Spectroscopy Facility. Reverse-phase HPLC purification and analysis were performed using an Agilent 1260 Infinity HPLC with Agilent 6120 Quadrapole mass spectrometer (Santa Clara, CA). PreQ1-TO derivatives were prepared and analyzed with an Agilent Zorbax SB-C18 semi-prep column (ID 9.4 x 250 mm, 5 µm, 80 Å) and an Agilent Zorbax eclipse plus C8 column, using a water/methanol gradient containing 0.1% TFA. Oligonucleotide HPLC analysis was carried out on a Phenomenex Clarity Oligo-MS analytical column (ID 2.1 x 50 mm, 2.6 µm, 100 Å) using a gradient of water containing 20% hexafluoroisopropanol (HFIP) and 0.1% TEA and methanol containing no additives. Fluorescence measurements were collected either on a JASCO FP-8500 fluorimeter (for spectral analysis and fluorescent turn-on assay) (Hachioji, Japan) or a Tecan Saphire-II plate reader (for kinetic analysis) (Tecan, Männedorf, Switzerland). Fluorescence microscopy images were acquired on an Axio Observer D1 inverted microscope (Carl Zeiss Microscopy GmbH, Germany) with a 20 x and 60 x and 1.42 NA air immersion objective and ORCA-ER camera (Hamamatsu, Japan) using the FLUOVIEW software package (Olympus, Japan). Fluorescent probes and proteins were excited with an argon laser at appropriate wavelengths. Images were subsequently analyzed and processed using ImageJ.
The synthesis of 3a-c was adopted from previously published protocol. 4 4-(methylthio)quinoline 5 and appropriate alkyl halide was dissolved in ACN or 1,4-dioxane. The mixture was stirred and heated overnight. The resulting solid was filtered and washed with diethylether. The solid was used directly without further purification.

Preparation of plasmid constructs
12.1 mCherry plasmid A synthetic gene block containing the sequence for mCherry was designed and ordered from IDT (Coralville, IA). Specifically, the gene was designed with the ECY-A1 hairpin downstream of the mCherry coding region within the 3' UTR. The geneblock was cloned into the mammalian expression vector pcDNA3 (Thermo Fisher, Waltham, MA) between cut sites BamHI and XhoI. DH5a competent cells (Life Technologies, Carlsbad, CA) were then transformed with the ligation product and screened against ampicillin on agar plates overnight. Colonies were selected and overgrown, and the overgrowth was subjected to DNA extraction with a QIAGEN Plasmid Maxi Kit (Qiagen, Venlo, Limburg Netherlands) and sequencing was performed to verify the inserted gene. 13. In Vitro transcription of mCherry mRNA 20 µL 600 ng/µL mCherry construct plasmid DNA was digested by 20 units XhoI in 500µL 1x CutSmart buffer in 37 °C warm water bath for 1h. Linearized DNA was then extracted with molecular biology grade phenol / chloroform / isoamyl alcohol (25:24:1) (Sigma-Aldrich, St. Louis, MO), and purified by ethanol precipitation. The DNA pellet was dissolved in RNAase free water. To set up in vitro transcription reaction, 15 µL of 333 ng/µL linearized DNA (5.0 µg), 44.1 µL RNAse free water, 10 µL of 10x T7-RNAP buffer, 20 µL NTPs (ATP, CTP, GTP, UTP, 25 mM each), additional 4 µL of 100 mM GTP, 0.2 µL of 2 U/µL inorganic PPase, 0.5 µL RNAse inhibitor murine, 1.2 µL of 12.5 µg/µL T7-RNAP were mixed together to give a total reaction volume of 100 µL. The transcription was run at 37 °C overnight. The reaction mixture was treated with 1.2 µL of 100 mM CaCl 2 followed by 1 µL 20 Units of Turbo DNAse (Life Technologies, Carlsbad, CA) at 37 °C for 30 min. The mixture was centrifuged at max speed (16,100 g) for 2 min. The supernatant was then added with 50 µL of 8 M LiCl and cooled to -20 °C for 40 min. The mixture was centrifuged again at 4 °C at max speed for 30 min. The supernatant was discarded, the pellet was allowed to dry at room temperature for 5 minutes by evaporation, and the pellet was subsequently dissolved in 100 µL RNAse free water to give a final mRNA concentration of 4800 ng/µL. Percentage labeling was calculated using the following equation: Percent labeling = (Area of TO-labeled ECY-A1) / (Area of All Peaks) x 100%. An estimation of 6f substrate kinetics with ECY-A1 was performed in a similar fashion to the previous published protocol. 8 A triplicate set of 20 µL reactions was analyzed using a Tecan Saphire-II plate reader in a Greiner 384 well flat bottom plate (VWR,. Each well was prepped with 10 µM ECY-A1 and a variable amount (from 100 µM to 5 nM in serial 1/3 dilutions) of 6f in TGT reaction buffer. The plate was covered and allowed to warm up to 37 °C for 10 minutes. TGT enzyme was added to a final concentration of 100 nM. The plate was immediately subjected to a fluorescence reading for the first kinetic time point at 37 °C. Each well was monitored with an excitation of 501 nm and emission of 531. The excitation bandwidth was set to 20 nm and the emission bandwidth was set to 5 nm. The gain was held fixed and the integration time was set to 1 second with 10 reads averaged to each data point collected. The sample was monitored for 20 minutes at 37 °C with a reading taken every 2 minutes. An ECYA1-PreQ1-TO calibration curve was used to convert fluorescence into concentration of product in each sample for each time point to achieve an estimation of the initial rate of each reaction. The initial rates were then plotted against the concentration of substrate to achieve a standard kinetic curve. Chinese Hamster Ovary (CHO) cells (CRL-9606, ATCC, Manassas, VA) were cultured in Ham's F-12K media (Life Technologies, Carlsbad, CA). CHO cells were plated at an initial density of 10,000 cells per well in a Nunc Lab-Tek 8 well chamber slide (Thermo Scientific, Waltham, MA). Cells were allowed to adhere overnight and washed with Opti-MEM media (Life Technologies, Carlsbad, CA) and subsequently transfected with 1200 ng of mCherry construct plasmid per well with 1.6 µL of Lipofectamine 2000 (Life Technologies, Carlsbad, CA) in Opti-MEM as per manufacture's protocol. Control wells lacking transfection were treated and washed with Opti-MEM in the absence of DNA and Lipofectamine. After overnight transfection, the cells were washed twice with F-12K media (200 µL each) and allowed to recover at 37 °C for 6 h. The cells were fixed with 100 µL of a 3.7% paraformaldehyde in PBS solution for 30 minutes at room temperature and then permeabilized by treatment with 100 µL of 0.1% Triton X-100 in HBSS buffer (Life Technologies, Carlsbad, CA) for 30 minutes at room temperature and washed 2 x 200 µL with HBSS buffer. Cells were then treated for 3 h at 37 °C with 100 µL of TGT reaction buffer containing 500 nM 6f either with or without 500 nM TGT enzyme added. After incubation, the cells were directly subjected to fluorescent microscopy imaging. TO probe 6f was excited with a 488 HeNe laser and mCherry fluorescent protein was excited with a 543nm HeNe laser. Images were analyzed and processed using ImageJ (NIH, rsbweb.nih.gov).

Verification of TGT expression in HeLa cells by immunofluorescent imaging
HeLa cells were first transfected in a similar manner to previous experiments. Cells were plated at 40,000 cells per well in an 8-well chambered lab-tek slide and allowed to adhere overnight. Cells were then washed and transfected with either mCherry plasmid or co-transfected with equal concentrations of mCherry and CMV-TGT plasmids using standard Lipofectamine protocols. After overnight transfection, cells were washed, fixed and permeabilized in a similar manner to prior imaging studies. Fixed cells were then blocked for 30 min. at room temperature with a PBS solution containing 1% BSA and 0.1% Tween 20 (Solution A) to block unspecific binding of antibodies. The cells were then incubated with diluted (1 µg/uL) 6x-His Tag monoclonal antibody, Clone HIS.H8 (# MA121315, Invitrogen) in Solution A overnight at 4 °C. Cells were subsequently washed 3x with PBS and incubated for 1 hour at r.t. in the dark with diluted (1 µg/uL) Donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor 488 (# A-21202, Invitrogen) in Solution A according to manufacturer's protocols for immunofluorescent imaging. Cells were washed 3x with PBS and imaged by confocal microscopy in a similar fashion to our RNA imaging protocols. Fig. S6. Varying TGT expression in HeLa cells. HeLa cells were co-transfected with a plasmid capable of expressing TGT along with the mCherry plasmid (top), or mCherry plasmid alone (bottom). After 18 h, the cells were fixed and labeled with a 6x-His mouse antibody overnight at 4 °C, washed, and subsequently labeled with a donkey anti-mouse Alexa-488 conjugated antibody for immunoflurorescent imaging. The secondary antibody was washed and the cells were imaged by confocal microscopy. Anti-6x His antibody should selectively label the His Tag bearing TGT protein (green) which was observable by strong staining of co-transfected cells, while cells expressing mCherry alone were not stained by the anti-His antibody. mCherry expression (red) was evident in mCherry transfected cells, however the expression level was reduced in co-transfected cells presumably due to coexpression with the TGT construct.
S23 21. Live cell labeling of mCherry mRNA with 6f via microinjection HeLa cells were cultured in DMEM media (Life Technologies, Carlsbad, CA) and were plated in a 50 mm glass bottom dish (Electron Microscopy Sciences, Hatfield, PA). Cells were allowed to adhere overnight and washed with Opti-MEM media (Life Technologies, Carlsbad, CA). A solution of 10 µL of 10 µM 6f and 10 µM TGT in 1x TGT buffer was loaded in a femtotip II capillary (Eppendorf, Hamburg, Germany). The mixture was injected in 50 individual cells using an Eppendorf Micromanipulator 5171 and an Eppendorf Microinjector 5242. The injection pressure was set to 1400 hPa and the hold pressure was set to 500 hPa. Control cells were injected with the same concentration of 6f with no TGT in 1x TGT buffer. The cells were incubated at 37 °C for 3 h and subjected to confocal fluorescence microscopy.  Fig. S5. mCherry construct transfected CHO cells were fixed, permeabilized, and treated with 2 µM 6f in the presence or absence of 0.5 µM TGT. Cells were incubated at 37 °C for 3 h and imaged using a fluorescence microscope. Similar fluorescent intensity was observed for both cells with and without the treatment of TGT. The high background staining suggests that the increased concentration of 6f is not a suitable for cellular mRNA imaging.