Flow cytometric detection of specific RNAs in native human cells with quenched autoligating FRET probes -- Data Supplement - HTML Page - 09938SuppText.htslp -- Proceedings of the National Academy of Sciences

Supporting Materials and Methods
Synthesis of Probes
. Oligonucleotides were synthesized with b -cyanoethyl phosphoroamidite chemistry, deprotected by 25% ammonia solution for 12 h, and purified by reverse-phase HPLC. The synthesis of the dabsyl linker phosphoroamidite for the 5' end of the electrophile probe was performed as described in ref. 1. The electrophile probes containing both a dabsyl linker and a fluorescein label were prepared following procedures described in ref. 1. For synthesis of electrophile oligonucleotides with 2'-O-methyl nucleotides, ultramild 2'-OMe Pac-phosphoroamidites (Glen Research, Sterling, VA) were used at each position except for the position labeled with fluorescein. To characterize an electrophilic probe produced by this procedure, we obtained MALDI-MS data for the b -actin probe 5'-(dabs-linker)-T(FAM)-AGCACAGCCTGGA-3'. The observed mass was 5778.7 (calculated value, 5777.9).

For synthesis of nucleophilic probes carrying a Cy5-label and a 3'-phosphorothioate group, we used the following procedure. Solid-supported synthesis began with a 3'-phosphate-ON controlled pore glass column (Glen Research). This column was sulfurized by a commercial reagent (Glen Research). Next, an asymmetric branching amidite, 5'-DMT-T-C6-O-Lev (Biosearch), was coupled. The oligonucleotide sequence then was added following standard procedures. The Cy5 dye was then introduced by the following procedure: after the final nucleotide was coupled with trityl on, the CPG column was treated with a buffered hydrazine solution (2). This was done by removing the column from the synthesizer and introducing the reagents with syringe. It was treated with 0.5 M hydrazine monohydrate in pyridine/acetic acid solution (2:1) for 15 min and washed three times with acetonitrile. Then, the column was placed back on the synthesizer, and Cy5 Linker Phosphoroamidite (Biosearch) was coupled. Deprotection and removal from the solid support was done by treatment with 0.5 M K₂CO₃ in methanol solution for 4 h. The probes were purified by reverse-phase HPLC (Econosil C18 22 × 250 mm, 10 m m, Alltech, eluting with 0.1 M triethylammonium acetate (pH 7.0)/acetonitrile). For preparation of nucleophile oligonucleotides with 2'-O-methyl nucleotides, ultramild 2'-OMe Pac-phosphoroamidites (Glen Research) were used at each position except for the 3'-terminal position and the position labeled with Cy5. To characterize a nucleophile probe produced by this procedure, we obtained MALDI-MS data for the b -actin probe (5'-CAGAGGCGT(Cy5)ACAGGG-ps-3'). The observed mass was 6051.8 (calculated value, 6111.4).

Autoligation Reactions in Solution.
Ligations were performed in 3 ml of Na•Pipes buffer (70 mM, pH 7.0) containing 10 mM MgCl₂, 50 m M DTT with target nucleic acid (500 nM), dabsyl FAM-labeled probe (500 nM), and Cy5-labeled phosphorothioate probe (500 nM) at 25°C for 6 h. Reactions were observed by fluorescence spectrometry (Fluorolog 3-11, Jobin Yvon-SPEX). FRET spectra were measured under the following conditions: excitation, 490 nm; slit, 3 nm. For time courses of quenched autoligating-FRET ligation reactions, fluorescence intensity was measured for 1 s at 1-min intervals: excitation, 490 nm; emission, 667 nm; slit, 3 nm each. For the time course analysis, target DNA was added 17 min after starting the monitoring of a reaction solution containing dabsyl-FAM-labeled probe and Cy5-labeled phosphorothioate probe. Cell Culture.
HL-60 cells (American Type Culture Collection) were grown in DMEM without phenol red and containing 10% FBS, 50 units/ml penicillin, and 50 m g/ml streptomycin. They were passaged in 75-cm² culture flasks (Falcon). Cells were maintained at 37°C in an atmosphere of 5% CO₂ at 1 × 10⁵ cells/ml and within 0~20 passages after purchasing from the supplier. Reversible Permeabilization by Streptolysin O (SLO).
SLO was purchased from Sigma-Aldrich. SLO was activated according to the procedure reported by Faria et al. (3). Briefly, SLO (1,000 units/ml) was incubated in Mg²⁺/Ca²⁺-free PBS buffer solution containing 10 mM DTT and 0.05% BSA at 37°C for 2 h. Small aliquots of activated SLO were stored at –20°C.

To introduce probes into cells, HL-60 cells were washed with Mg²⁺/Ca²⁺-free PBS twice and then incubated with 300 m l of Mg²⁺/Ca²⁺-free PBS buffer solution containing dabsyl-FAM-labeled probe (200 nM), Cy5-labeled-phosphorothioate probe (200 nM), calf thymus DNA (1 m g/ml), and Hoechst 33342 stain (0.2 m g/ml) (Molecular Probes). SLO then was added (30 units/ml final concentration). After 30 min, cells were resealed by the addition of 1 ml of DMEM containing CaCl₂ (0.2 g/liter) and incubated for 1 h at 37°C.

Flow Cytometric Analysis.
The live cell suspension was directly analyzed without any washing step with a FACScan instrument (Becton Dickinson). FRET signals were observed under the following conditions: excitation by argon laser, 488 nm; emission, 660~675 nm. Forward angle light scatter (FSC), side angle light scatter (SSC), and fluorescence (FRET) data were recorded, and for each measurement, 50,000 events were stored. Data were analyzed with the FLOWJO program (Version 4.6.2, Tree Star, Ashland, OR). FRET intensity was determined as the median of FRET value of single cells lying in a gate that were defined in a FSC vs. SSC dot plot, in which almost 95% cells were gated. FRET signals were corrected by subtraction of background fluorescence of negative control (no probe). Confocal Microscope Imaging.
Cell suspensions were concentrated to 20-m l volumes. After reaction with QUAL-FRET probes for 3 h, live cells were directly spotted onto a glass slide without any washing step and covered with a glass cover slide. Fluorescence images were obtained through a Zeiss LSM 510 confocal laser-scanning microscope equipped with Plan-Apo 63´ objective lens. Microscope settings were as follows: frame size, 1024 × 1024; data depth, 12 bits; scan speed, 1.76 m s ´ 1.76 m s; scan mode, 3 channels (bright field, Hoechst33342, Cy5). Hoechst 33342 imaging: excitation by Ti:sapphire laser, 780~800 nm (two photon system); emission, 435~485 nm BP filter; pinhole, 852 m m. FRET Cy5 imaging: excitation by argon laser, 488 nm; emission, 650 LP filter; pinhole, 886 m m. The resulting raw data were analyzed by LSM image browser (Zeiss).

1. Abe, H. & Kool, E. T. (2004) J. Am. Chem. Soc. 126, 13980-13986.

2. Xu, Y. & Kool, E. T. (1999) Nucleic Acids Res. 27, 875-881.

3. Inoue, H., Hayase, Y., Imura, A., Iwai, S., Miura, K. & Ohtsuka, E. (1987) Nucleic Acids Res. 15, 6131-6148.