Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy

Cell culture, 5EU and EdU labeling, transfection

U2OS cells were cultured on 35 mm MatTek dishes (MatTek Corp) in DMEM supplemented with 10% FBS at 5% CO2. On reaching 70% confluency and two days before the experiment, cells are transfected with 0.5 μg of DNA with lipofectamine 3000 (ThermoFisher Scientific) following the manufacturer protocol. If needed, cells are incubated overnight in culture medium with 5 μM of 5-ethynyl 2′-deoxyuridine (EdU, #1149, Click Chemistry Tools) diluted from a 10 mM stock in DMSO. For 5-EU  (5-ethynyluridine) incorporation, 200 mM 5-EU (#1261, Click Chemistry Tools) in DMSO is diluted to 1 mM in culture medium and incubated with cells overnight.

DNA constructs

For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-6²⁵ and substituted EGFP in pHaloTag-EGFP (addgene #86629)²⁶ through the In-Fusion cloning method. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)²⁷ and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method.

Synthesis of TMR-PROMPT and TMR-PEG4-PROMPT (Fig. S2)

NH₂-cys(S-Trt)-CO-NH-(CH₂)₃-N₃ (2)

Fmoc-NH-cys(S-Trt)-OH, (1) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 µL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing. The reaction mix turned yellow and LC–MS revealed complete reaction in 30 min; ES-MS (m/z) [M + Na]⁺ for C₄₀H₃₇N₅NaO₃S, 690.25; found 689.3. Piperidine (20 μL, 0.2 mmol) was added and the solution evaporated under high vacuum after 1 h, dissolved in DMSO, separated by RP-HPLC and lyophilized to give 2 as a white solid. Yield, 14.3 mg, 76%. ES-MS (m/z) [M]⁺, [M + Na]⁺ for C₂₅H₂₇N₅OS, 446.2, 468.2; found 446.1, 468.1.

5(6)-TMR-CONH-cys(SH)-CO-NH-(CH₂)₃-N₃ (4)

5(6)-Carboxytetramethylrhodamine, 5(6)-TMR-CO₂H (1.35 mg, 3.14 μmol, Novabiochem) and TSTU (1.3 mg, 4.4 μmol) were dissolved in dry DMSO (25 μL) with TEA (0.96 μL, 6.9 μmol) and kept at room temperature. Reaction was complete in 30 min (by LC–MS) and then added to a solution of NH₂-cys(S-Trt)-CO–NH-(CH₂)₃-N₃ (2) (2.0 mg, 3.6 μmol) in DMSO (10 μL) with NMM (1 μL, 9.1 μmol) and kept at room temperature overnight when LC–MS revealed complete reaction. After acidification with HOAc (2 μL), the desired product, 3 was isolated by RP-HPLC and lyophilized to a red solid. Yield, 2.0 mg (74%) ES-MS (m/z) [M]⁺ for C₅₀H₄₈N₇O₅S, 858.3; found 858.3. The trityl group was removed by dissolving the product (1.89 mg, 2.2 μmol) in TFA:H₂O:Triisopropylsilane:Ethanedithiol (92.5:2.5:2.5:2.5 v/v, 0.5 mL) for 30 min, evaporation under high vacuum, purification by RP-HPLC and lyophilization to give 4 as a red solid. Yield, 0.9 mg (66%) ES-MS (m/z) [M]⁺ for C₃₁H₃₄N₇O₅S, 616.2; found 616.1.

SPDP-HaloTag linker

HaloTag linker amine (²⁸ (3.0 mg, 13.5 μmol) and SPDP (4.7 mg, 15 μmol) were dissolved in dry DMSO (50 μL) and NMM (3.3 μL, 30 μmol) added. LC–MS revealed reaction was complete after overnight when the reaction mixture was neutralized with HOAc (5 μL) and the product was purified by RP-HLPC (and lyophilized to a colorless oil. Yield, 3 mg (54%) ES-MS (m/z) [M]⁺ for C₁₈H₃₀ClN₂O₃S₂, 421.1; found 421.1.

TMR-PROMPT; 5(6)-TMR-CONH-cys(S-S-(CH₂)₂CONH-HaloTag ligand)-CO-NH-(CH₂)₃-N₃ (5)

A solution of 5(6)-TMR-CONH-cys(SH)-CO-NH-(CH₂)₃-N₃ (4) in DMSO (100 μL, 6.25 mM measured by absorbance in 0.1 M HCl in 95% ethanol using ε_max 95,000 M⁻¹ cm⁻¹ at 554 nm, 0.626 μmol) was mixed with SPDP-HaloTag linker (20 μL, 49 mM in dry DMSO, 0.98 μmol) and NMM (1 μL, 10 μmol) added. After 1 h, HOAc (5 μL) was added and the desired product, (5) purified by RP-HPLC to give a colorless oil. Yield, 0.42 mg (72%) ES-MS (m/z) [M]⁺ for C₄₄H₅₈ClN₈O₈S_2, 925.4; found 925.4.

SPDP-PEG4-HaloTag linker

Solutions of HaloTag linker amine²⁸ (3.0 mg, 4.5 μmol,) in dry DMSO (90 μL) and SPDP-PEG4-NHS (2.5 mg, 4.5 μmol, Quanta Biodesign) in dry DMSO (90 μL) were mixed and NMM (1 μL, 10 μmol) added. LC–MS revealed reaction was complete after 4 h and the product was used without further purification. ES-MS (m/z) [M]⁺ for C₂₉H₅₁ClN₃O₈S₂, 668.3; found 668.3.

TMR-PEG4-PROMPT; 5(6)-TMR-CONH-cys(S-S-(CH₂)₂CONH-PEG4-HaloTag ligand)-CO-NH-(CH₂)₃-N₃ (6)

5(6)-TMR-CONH-cys(SH)-CO-NH-(CH₂)₃-N₃ (4) (3 mL of 0.25 mM in DMSO; measured as above) was added to the solution of SPDP-PEG4-HaloTag Linker (15 μL of 50 mM, 0.75 μmol) and NMM (15 μL, 150 μmol) added. After 2 h, LC–MS indicated reaction was complete, HOAc (50 μL) was added, the product, (6) purified by RP-HPLC and lyophilized. Yield, 0.7 μmol (by absorbance in 0.1 M HCl in 95% ethanol using ε_max 95,000 M⁻¹ cm⁻¹ at 554 nm) after dissolving in dry DMSO (100 μL). ES-MS (m/z) [M]⁺ for C₅₅H₇₉ClN₉O₁₃S₂, 1172.5; found 1172.4.

Synthesis of JF525-PROMPT and JF590-PROMPT (Fig. S3)

Fmoc-NH-cys(SH)-CO-NH-(CH₂)₃-N₃ (7)

Fmoc-NH-cys(S-Trt)-OH (1) (59 mg, 100 μmol) and HATU (42 mg, 110 μmol) were dissolved in dry DMF (200 µL) in a plastic screw cap tube and 3-azidopropyl-1-amine (11 μL, 110 μmol, Click Chemistry Tools) followed by DIEA (38 μL, 220 μmol) were added with mixing. The reaction mix turned yellow and LC–MS revealed complete reaction in 30 min; ES-MS (m/z) [M + Na]⁺ for C₄₀H₃₇N₅NaO₃S, 690.3; found 690.2. The reaction mixture was evaporated to yellow oil and trityl group removed by dissolving in TFA-H₂O-Triisopropylsilane-Ethanedithiol (92.5/2.5/2.5/2.5 v/v, 1 mL) and kept at room temperature for 2 h. Following evaporation to an oily solid, the desired product was purified by RP-HPLC, and lyophilized to give (7) a white solid. ES-MS (m/z) [M]⁺ for C₂₁H₂₄N₅O₃S, 426.2; found 426.1.

NH₂-cys(S-S-(CH₂)₂CONH-HaloTag ligand)-CO-NH-(CH₂)₃-N₃

Fmoc-NH-cys(SH)-CO-NH-(CH₂)₃-N₃ (7) (1.25 μmol, 50 μL of 25 mM solution in dry DMSO) was mixed with SPDP-HaloTag linker (1.25 μmol, 25 μL of 50 mM solution in dry DMSO) and NMM (2.5 μL, 25 μmol) added. LC–MS revealed complete reaction after 20 min, ES-MS (m/z) [M]⁺ for C₃₄H₄₈ClN₆O₆S_2, 735.3; found 735.3. Piperidine (20 μL) was added and the solution evaporated after 5 min. The residue was dissolved in DMSO and HOAc (5 μL), product 8 isolated by RP-HPLC, lyophilized and dissolved in dry DMSO (75 μL). ES-MS (m/z) [M]⁺ for C₁₉H₃₈ClN₆O₄S₂, 513.2; found 513.2.

JF570-PROMPT; Janelia Fluor 570-CONH-cys(S-S-(CH₂)₂CONH-HaloTag ligand)-CO-NH-(CH₂)₃-N₃ (9)

The product, 8 from the previous step was reacted with JF570-NHS ester (0.4 μmol, 8 μL of 50 mM solution in dry DMSO) and NMM (10 μmol, 1 μL) for 2 days at room temp, the product 9 isolated by RP-HPLC, lyophilized and dissolved in DMSO (100 μL) to give a 1.4 mM solution, measured by ε_max 100,000 M⁻¹ cm⁻¹ in 0.1 M HCl in 95% ethanol at 574 nm. ES-MS (m/z) [M]⁺ for C₄₆H₅₈ClN₈O₇S₃, 965.3; found 965.3.

JF525-PROMPT; Janelia Fluor 525-CONH-cys(S-S-(CH₂)₂CONH-HaloTag ligand)-CO-NH-(CH₂)₃-N₃ (10)

Prepared as for JF570-PROMPT. Measured by ε_max 80,000 M⁻¹ cm⁻¹ in 0.1 M HCl in 95% ethanol at 532 nm. ES-MS (m/z) [M]⁺ for C₄₆H₅₄ClF₄N₈O₈S₂⁺,1021.3; found 1021.3.

Reaction with HaloTag protein

JF525-PROMPT or JF570-PROMPT (0.5 μL of 1 mM solution in DMSO) was added to HaloTag protein (2.5 μL of 100 μM solution in PBS) diluted in 100 mM Na·MOPS pH 7.2, and kept at room temperature for 2 h. Acetic acid (1 μL) was added and analyzed by LC–MS using a PLRP-S 1000A column (8 μm, 50 × 2.1 mm, Agilent) eluting with linear 20–60% ACN-water with constant TFA (0.05%) in 16 min.

JF570-PROMPT: ES-MS (m/z) [M]⁺ for adduct with HaloTag protein (loss of HCl), C₄₆H₅₇N₈O₇S₃, (965.3–35.98) 929.3; for deconvoluted masses of JF570-PROMPT:HTP complex, 35,729.0 minus HTP, 34,798.6; found 930.4.

JF525-PROMPT: ES-MS (m/z) [M]⁺ for adduct with HaloTag protein (loss of HCl), C₄₆H₅₃F₄N₈O₈S₂, 985.3; for deconvoluted masses of JF525-PROMPT:HTP, 35,783.5 minus HTP, 34,798.6, found 984.9.

PROMPT method

After EdU or 5EU labeling, transfected cells are washed with culture medium and fixed for 2 min with room temperature fixative (4% EM grade paraformaldehyde (Electron Microscopy Sciences #19202) + 0.1% EM grade glutaraldehyde (Ted Pella, #18426) in 0.1 M Hepes (Sigma-Aldrich #H3375) pH7.4 + 2 mM CaCl₂) followed by a 1-h incubation with 4 °C fixative. Next, the cells are washed three times with PBS and incubated for 10 min with PBS + 10 mM glycine. The cells are then incubated for 30 min with 200 nM of PROMPT probe in PBS + 0.1% BSA (Sigma-Aldrich #A8022) + 0.1% saponin (Sigma-Aldrich #S4521), then washed five times with PBS + 0.1%BSA + 0.1%saponin, and five times with PBS. For the click chemistry step, the cells are incubated for 2 times 30 min with 50 mM Hepes pH7.4 + 100 mM NaCl + 2 mM CuSO₄ + 10 mM of freshly prepared sodium ascorbate (Sigma-Aldrich #A7631). The reaction is terminated with ten washes of PBS. For the disulfide bond reduction, cells are incubated three times 10 min with freshly prepared 10 mM DTT (Sigma-Aldrich #D9779) in PBS. Released photosensitizers are removed with five washes of PBS + 0.1%BSA + 0.1%saponin and five washes with PBS.

Imaging and photooxidation

If not for EM, cells were imaged in PBS with an inverted confocal microscope Leica SPEII. Within the same experimental set, which includes triplicates, the imaging parameters (laser power, gain, and offset) are adjusted on the brightest samples/conditions for optimal use of the microscope detector’s dynamic range. For photooxidation, cells were imaged in 2.3 mM DAB (Sigma-Aldrich #D8001) in 0.1 M cacodylate (Sigma-Aldrich #C0250) buffer pH 7.4 with the same microscope. DAB was photo-oxidized by JF570-PROMPT under illumination with a 63× objective for 12 min using the 575/25 line (310 mW) of a Spectra 7 LED light source (Lumencor). Then samples were washed with 0.1 M cacodylate buffer pH 7.4 and processed for EM.

Image analysis

Fluorescence intensities were measured using ImageJ. Mean intensities were calculated from more than 30 cells from 3 independent experiments. In the figures, the brightness and contrast are optimized on the brightest sample/condition and applied to all the images displayed in the same experiment.

Resin embedding, sectioning, and TEM imaging

After photo-oxidation, samples were post-fixed for 30 min with 1% osmium (Electron Microscopy Sciences #19150) + 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer pH 7.4 at 4 °C. Then the samples were dehydrated in 1-min successive 4 °C baths of increasing ethanol concentration (20%, 50%, 70%, 90%, 3 × 100%), and incubated for 1-h in 50%ethanol/50% durcupan resin (Sigma-Aldrich #44611, #44612, #44613, #44614). Samples were then incubated 3 × 2-h in 100% durcupan mixture, and the resin was polymerized for 48 h at 60 °C. At the photo-oxidized areas, blocks of hardened resin were cut off from the dish and mounted onto dummy blocks using glue. The glass was popped up by water infiltration, and the blocks were trimmed around the cells of interest to shape a block face of approximatively 400 μm wide and 200 μm tall. Block faces were imaged with an upright light microscope at 10X to facilitate correlation. Ribbons of 70 nm thick sections were produced from these blocks and collected on glow-discharged formvar coated slot grids (Electron Microscopy Sciences #FF2021-Cu-ET). Areas of interest were imaged at 120 keV with an FEI Tecnai Spirit transmission electron microscope.

Multi tilt axis electron tomography

For electron tomography, 250 nm thick sections were produced and collected onto Luxel grids (Luxel, #LuxFilmTEM C-S-M-L), then incubated for 20 s with 20% 10 nm gold fiducials (Ted Pella#15703-20) + 0.05% BSA in water, blotted, and dry. The grids with sections were then imaged with an upright light microscope at 10×. The grids were loaded onto a rotation holder (Fischione Instruments, model 2040). After correlation, a four tilt-axis series (one tilt-series: − 60° to + 60°, one image every 0.25°) at different orientation (0°, 45°, 90°, 135°) of the region of interest was acquired using an FEI Titan Halo operated at 300 keV and a DE64 camera, while controlled by SerialEM²⁹. The entire set of 1936 acquired images were aligned and reconstructed using TxBR package^{30, 31}.