Experimental Example: Staining of G4 DNA and α-synuclein (αSyn) protein aggregates

The formation of protein–DNA aggregates was assessed using α-synuclein (αSyn) protein combined with either MycG4 (G4 DNA) or dsDNA-6 (dsDNA). Samples were stained with G-Quadruplex Dye and thioflavin T.

Preparation of a working solution

One (1) µl of G-Quadruplex Dye was diluted with 9 µl of PBS, and 1 µl of 10 mmol/l Thioflavin T DMSO solution was diluted with 99 µl of PBS.

*Thioflavin T was used in the (LL03) LLPS Characterization-dye Set.

Preparation of PEG8000 solution

A 40 % (w/v) PEG8000 solution were prepared with PBS. 

*PEG8000 was used in the (LL02) LLPS Forming Condition Screening Kit.

Preparation of DNA solution

Two 0.5-ml microtubes were prepared, and 44.3 µl of PBS was added to each tube; 1.0 µl of each 100 µmol/l DNA solution was added and mixed. 

Preparation, staining, and observation of αSyn aggregates

1. Four point five three (4.53) µl of DNA solution was added to each 0.5 ml microtube.
2. One (1) µl of G-Quadruplex Dye and Thioflavin T working solution was added.
3. Zero point seven two (0.72) µl of αSyn solution（SPR-321, StressMarq Biosciences）was added to each 0.5 ml microtube.
4. Three point seven five (3.75) µl of 40 % PEG8000 solution was added to each 0.5 ml microtube ; 20 pipetting cycles were performed for each 0.5 ml microtube.
   
   *Because of its high viscosity, the PEG8000 solution should be added slowly using reverse pipetting.
   *Manual pipetting is strongly recommended to avoid the foaming that tends to occur when using electric pipettes.
   ​*After adding the PEG8000 solution, promptly pipette to prevent protein aggregation if left standing for an extended period.

5. Two (2) µl of sample solution was added to a glass slide with double-sided tape with a hole. The glass slides were covered with a cover glass and incubated at 37°C for 18 days, protected from light. After incubation, the samples were observed under a fluorescence microscope. 

Fig4. Fluorescence imaging of aggregates and G4 of each oligo DNA and αSyn protein

Bright-field microscopy and thioflavin T staining revealed that aggregates formed only in the condition where MycG4 (G4 DNA) was present (middle row). Additionally, staining with G-Quadruplex Dye confirmed the accumulation of G4 DNA within the aggregates.

Experimental Example: Fluorescence imaging of dsDNA and G4 in living and fixed cells

- Assay for living cells -

1.     HeLa cells (2.0 x 10⁴ cells/well, MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin) were seeded on a μ-slide 8 well plate (ibidi) and cultured at 37°C overnight in a 5% CO₂ incubator.
2.     After the culture medium was removed, the cells were washed once with 200 μl of assay buffer (1x).
3.     After the supernatant was removed, a working solution (200 μl) was added to the cells.
4.     The cells were incubated at 37°C in a 5% CO₂ incubator for 10 min.
5.     After the supernatant was removed, the cells were washed with 200 μl of Opti-MEM twice.
6.     Opti-MEM (200 μl) was added, and the cells were observed by fluorescence microscopy.

- Assay for fixed cells -

1.     HeLa cells (2.0 x 10⁴ cells/well, MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin) were seeded on a μ-slide 8 well plate (ibidi) and cultured at 37°C overnight in a 5% CO₂ incubator.
2.     After the culture medium was removed, the cells were washed with 200 μl of PBS once.
3.     One hundred (100) μl of 4% paraformaldehyde (PFA) / PBS solution was added to the cells and incubated at room temperature for 10 minutes.
4.     The 4% PFA / PBS solution was removed, and the cells were washed with 200 μl of PBS Two times.
5.     The cells were washed once with 200 μl of assay buffer (1x).
6.     Two hundred (200) μl Working solution was added to the cells and incubated at 37°C for 10 minutes.
7.     The cells were observed by fluorescence microscopy.

Figure 1. Fluorescence signals from HeLa cells detected with a microscope

Experimental Example: Detection of dsDNA and G4 in DNA oligonucleotide using G-Quadruplex Dye

Preparation of G-Quadruplex Dye solution

G-Quadruplex Dye (30 μl) was diluted by adding 20 μl of DMSO.

Preparation of 100 μmol/l DNA solution

dsDNA-6, dsDNA-4, c-Myc, and MycG4 (all from Macrogen) were dissolved in 50 mmol/l Tris–HCl buffer containing 100 mmol/l KCl to prepare a 100 µmol/l DNA solution.

Table 4. Oligonucleotides sequences

Staining and measurement of DNA

1. One hundred (100) µl of 250 mmol/l Tris-HCl solution was added to each 1.5 ml microtube.
2. One hundred (100) µl of 500 mmol/l KCl solution was added to each tube.
3. Fifty (50) µl of G-Quadruplex Dye solution was added to each tube.
4. Two hundred and twenty (220) µl of ddH₂O was added to each tube.
5. Thirty (30) µl of 100 μmol/l DNA solution was added to each tube.
6. 150 µl of sample solution was added to each well of a 96-well black clear-bottom microplate (IWAKI, 5882-096), and the fluorescent signal was measured using a plate reader.

Figure 2. Detection of dsDNA and G4 in each DNA oligonucleotide

Each DNA oligonucleotide sample was stained with G-Quadruplex Dye. The results showed that when excited at 470 nm, dsDNA-6 and dsDNA-4, which have a dsDNA structure, exhibited high fluorescence intensity. When excited at 570 nm, c-Myc and MycG4, which contain abundant G4 DNA structures, exhibited high fluorescence intensity.

Experimental Example: Fluorescence imaging of the intracellular G4 in A549 cells treated with Pyridostatin (PDS)

1. A549 cells (2.0 x 10⁴ cells/well, MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin) were seeded on a μ-slide 8 well plate (ibidi) and cultured at 37°C overnight in a 5% CO₂ incubator.
2. After the culture medium was removed, the cells were washed once with 200 μl of assay buffer (1x).
3. After the supernatant was removed, a working solution (200 μl) was added to the cells.
4. The cells were incubated at 37°C in a 5% CO₂ incubator for 10 min.
5. After the supernatant was removed, the cells were washed with 200 μl of Opti-MEM twice.
6. Twenty (20) μmol/l PDS diluted in Opti-MEM was added, and the cells were incubated at 37°C for 0–90 minutes in a 5% CO₂ incubator, and then observed under fluorescence microscopy.

Figure 3. Fluorescence imaging of PDS-treated A549 cells

The formation of G4 structures in live cells was detected using G-Quadruplex Dye in the presence of PDS, a G4-stabilizing compound. Fluorescence microscopy images were captured every 15 min. Over time, fluorescence at 700 nm increased, confirming the accumulation of G4 structures in the cells.

Experimental Example: Flow cytometric detection of the intracellular G4 in A549 cells treated with Pyridostatin (PDS)

1. A549 cells (1.0 x 10⁵ cells/well, MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin) were seeded on a6 well plate  and cultured at 37°C 48 hours in a 5% CO₂ incubator.
2. After the culture medium was removed, the cells were washed once with 2 ml of assay buffer (1x).
3. After the supernatant was removed, a working solution (2 ml) was added to the cells.
4. The cells were incubated at 37°C in a 5% CO₂ incubator for 10 min.
5. After the supernatant was removed, the cells were washed with 2 ml of Opti-MEM twice.
6. 20 μmol/l PDS diluted in Opti-MEM was added, and the cells were incubated at 37°C for 90 minutes in a 5% CO₂ incubator, and then signals were detected by flow cytometry.

The formation of G4 structures in live cells was detected by flow cytometry using G-Quadruplex Dye in the presence of PDS, a G4-stabilizing compound. After PDS stimulation, fluorescence signals from G-Quadruplex Dye at 700 nm increased, confirming the accumulation of G4 structures in the cells.