8. Methods
8.5. SELEX
8.5.1. Cell-SELEX
8.5.1.1. SELEX targeting Nav1.5-HEK293 and Nav1.6-HEK293 using D3-library The cells were thawed as described in section 8.4.2. After cultivating for 10 days, the cells were used for the selection. The expression of the VGSCs was confirmed via the patch-clamp technique in the cooperation work with the group or Prof. Heinz Beck (Universität Bonn). In a 12.5cm² or 25cm² tissue culture flask, 0.5 ∙ 106 or 1 ∙ 106 cells were seeded and incubated for 24h at 37°C and 5% CO2. Then the cells were washed twice with 10mL DMEM and then incubated with the DNA-library in binding buffer (DMEM, 10%FCS, MEM Non-Essential Amino Acids (NEAA), and sodium pyruvate) for 30 min at 37°C and 5% CO2. Afterward, the supernatant was discard and the cells were washed with DMEM. Finally, the cells were scraped in 500 µL ddH2O and transferred to a 2 mL tube. The bound DNA sequences were recovered by heating for 5 min to 95°C followed by phenol/chloroform extraction (section 8.1.3.2). The purified DNA was used as the template in the PCR. The detailed selection conditions are summarized in Table 8.9.
Table 8.9 Summary of cell-SELEX targeting Nav1.5-HEK293 and Nav1.6-HEK293 conditions.
SELEX cycle
D3-DNA-library HEK293 cells Nav1.x-HEK293 cells Wash
at 37°C, 5%CO2
1 1 nmol 6.9 ∙ 106 4.8 ∙ 106 2x 60 sec
2 500 pmol 2x 6.9 ∙ 106 4.8 ∙ 106 3x 60 sec
3 200 pmol 2x 6.9 ∙ 106 4.8 ∙ 106 4x 60 sec
4 100 pmol 3x 6.9 ∙ 106 4.8 ∙ 106 5x 60 sec
5 100 pmol 3x 6.9 ∙ 106 4.8 ∙ 106 6x 60 sec
6 100 pmol 3x 6.9 ∙ 106 4.8 ∙ 106 7x 60 sec
7 100 pmol 3x 6.9 ∙ 106 4.8 ∙ 106 7x 60 sec
8 100 pmol 4x 6.9 ∙ 106 4.8 ∙ 106 7x 60 sec
9 100 pmol 5x 6.9 ∙ 106 4.8 ∙ 106 7x 60 sec
10 100 pmol 5x 6.9 ∙ 106 4.8 ∙ 106 7x 60 sec
8.5.1.2. SELEX targeting Nav1.1-HEK293 and Nav1.2-HEK293 using D3-library The selection was carried out in a selection buffer (DMEM, 10% FCS, NEAA, sodium pyruvate, and 1mg/mL salmon sperm DNA). As a target, the Nav1.1-HEK293 or Nav1.2-HEK293 cells were used.
The D3-DNA library was incubated with the cells for 30 min at 37°C and 5% CO2. From the fourth selection cycle onwards, a counter selection step was introduced. The library was incubated first with the HEK293 cells in a T75 flask, followed by further incubations with fresh HEK293 cells, and finally the incubation with Nav1.x-HEK293 cells for 30 min at 37°C and 5% CO2. Afterward, the supernatant was discard and the cells were washed with DMEM. Finally, the cells were scraped in 500 µL ddH2O and transferred to a 2 mL tube. The bound DNA sequences were recovered by heating for 5 min to 95°C followed by phenol/chloroform extraction (section 8.1.3.2). The purified DNA was used as the template in the PCR. The detailed selection conditions are summarized in Table 8.10.
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Table 8.10 Summary of cell-SELEX targeting Nav1.1-HEK293 and Nav1.2-HEK293 conditions.
SELEX cycle
D3-DNA-library
HEK293 cells in T75 flask
Incubation time for each HEK293[min]
Nav1.x-HEK293 cells in T25 flask
Wash at 37°C, 5%CO2
1 500 pmol - 30 2 ∙ 106 1x 10 sec
1x 120 sec
2 20 pmol - 30 4 ∙ 106 1x 10 sec
1x 120 sec
3 20 pmol - 30 4 ∙ 106 1x 30 sec
2x 120 sec
4 20 pmol 5x 5 ∙ 106 30 3 ∙ 106 1x 30 sec
2x 120 sec
5 5 pmol 5x 4 ∙ 106 30 3 ∙ 106 1x 30 sec
2x 120 sec
6 0.05 pmol 5x 4 ∙ 106 60 3 ∙ 106 1x 30 sec
2x 120 sec
7 5 fmol 7x 4 ∙ 106 3x 60
4x 30 3 ∙ 106 1x 30 sec
2x 120 sec
8 1 fmol 7x 4 ∙ 106 3x 60
4x 30 3 ∙ 106 1x 30 sec
2x 120 sec
9 1 fmol 10x 4 ∙ 106 1x 60
9x 30 3 ∙ 106 1x 30 sec
2x 120 sec
8.5.1.3. Click–SELEX targeting Nav1.6-HEK293 using TTX-Elution
The selection was carried out in a selection buffer (DMEM, 10% FCS, NEAA, sodium pyruvate, and 1mg/mL salmon sperm DNA). As a target, the Nav1.6-HEK293 cells were used. The FT2 library was functionalized with guanidine azide (11) like in section 8.2.1 and 8.2.2 described. This functionalized library was incubated first with the HEK293 and finally the incubation with Nav1.6-HEK293 cells for 30 min at 37°C and 5% CO2. Afterward, the supernatant was discard and the cells were washed with DMEM. Finally, the elution buffer (D-PBS with 5mM MgCl2 and 1 mM CaCl2, 20 µM TTX) was added to the cells and incubated for 5 min at RT. The supernatant was transferred to an Amicon 10K column and centrifuged for 5 min at 1400 rpm, the flowthrough was discard and the column was washed 4x 500 µL ddH2O. Then the column was flipped into a fresh tube and centrifuged for 2 min at 1000 rpm. The recovered solution was used as the template in the PCR. The detailed selection conditions are summarized in Table 8.11.
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Table 8.11 Summary conditions of click-SELEX targeting Nav1.6-HEK293.
SELEX cycle
clicked-library
HEK293 cells in T75 flask
Nav1.6-HEK293 cells in T75 flask
Wash at RT
1 500 pmol 15 ∙ 106 15 ∙ 106 2x 30 sec
2 150 pmol 15 ∙ 106 15 ∙ 106 2x 30 sec
3 50 pmol 2x 15 ∙ 106 15 ∙ 106
1x 30 sec 1x 5 min 1x 30 sec
4 30 pmol 2x 15 ∙ 106 15 ∙ 106
1x 30 sec 1x 5 min 1x 30 sec
5 30 pmol 3x 15 ∙ 106 15 ∙ 106
1x 30 sec 1x 5 min 1x 30 sec
6 25 pmol 3x 15 ∙ 106 15 ∙ 106
1x 30 sec 1x 5 min 1x 30 sec
7 25 pmol 4x 15 ∙ 106 15 ∙ 106
1x 30 sec 1x 5 min 1x 30 sec
8.5.1.4. Click–SELEX targeting Nav1.6-HEK293
The selection was carried out in a selection buffer (DMEM, 10% FCS, NEAA, sodium pyruvate, 1mg/mL salmon sperm DNA, and 500 pmol functionalized click-competitor). As a target, the Nav 1.6-HEK293 cells were used. The FT2 library and the click-competitor was functionalized with indole azide (1) or guanidine azide (11) like in section 8.2.1 and 8.2.2 described. This functionalized library was incubated first with the HEK293 and finally the incubation with Nav1.6-HEK293 cells for 30 min at 37°C and 5% CO2. Afterward, the supernatant was discard and the cells were washed with DMEM.
Finally, the cells were scraped in 500 µL ddH2O and transferred to a 2 mL tube. The bound DNA sequences were recovered by heating for 5 min to 95°C followed by phenol/chloroform extraction (section 8.1.3.2). The purified DNA was used as the template in the PCR. The detailed selection conditions are summarized in Table 8.12.
After eight selection cycle, a branch point was introduced. The enriched library was aliquoted in two samples. With one sample the selection was continued like before, and with the other sample, the target molecule was switched, meaning that for the counter selection step the Nav1.6-HEK293 cells were used and as target cell the HEK293 cells.
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Table 8.12 Summary conditions of click-SELEX targeting Nav1.6-HEK293 using guanidine (11) and indole azides (1).
SELEX cycle
clicked-library
HEK293 cells in T75 flask
Nav1.6-HEK293 cells in T25 flask
Incubation time [min]
Wash at RT
1 500 pmol 10 ∙ 106 4.5 ∙ 106 30 2x 30 sec
2 40 pmol 10 ∙ 106 4.5 ∙ 106 30 1x 30 sec, 1x 3 min,
1x 30 sec
3 80 pmol 10 ∙ 106 4.5 ∙ 106 25 1x 30 sec, 1x 3 min,
1x 30 sec
4 50 pmol 10 ∙ 106 4.5 ∙ 106 25 1x 30 sec, 1x 3 min,
1x 30 sec
5 50 pmol 10 ∙ 106 4.5 ∙ 106 20 1x 30 sec, 1x 3 min,
1x 30 sec
6 50 pmol 10 ∙ 106 4.5 ∙ 106 20 1x 30 sec, 1x 5 min,
1x 30 sec
7 50 pmol 10 ∙ 106 4.5 ∙ 106 15 1x 30 sec, 1x 5 min,
1x 30 sec
8 30 pmol 10 ∙ 106 4.5 ∙ 106 15 1x 30 sec, 1x 5 min,
1x 30 sec
9 20 pmol 10 ∙ 106 4.5 ∙ 106 15 1x 30 sec, 1x 5 min,
2x 30 sec
10 20 pmol 10 ∙ 106 4.5 ∙ 106 15 1x 30 sec, 1x 5 min,
2x 30 sec
11 20 pmol 10 ∙ 106 4.5 ∙ 106 15 1x 30 sec, 1x 5 min,
2x 30 sec
12 20 pmol 10 ∙ 106 4.5 ∙ 106 15 1x 30 sec, 1x 5 min,
2x 30 sec
8.5.1.5. Click–SELEX targeting GluR1-HEK293
The selection was carried out in a selection buffer (DMEM, 10% FCS, 1mg/mL salmon sperm DNA, and 500 pmol functionalized click-competitor). As a target, the GluR1-HEK293 cells were used. The OW1 library and the click-competitor was functionalized with indole azide (1), benzyl azide (2), or guanidine azide (11) like in section 8.2.1 and 8.2.2 described. This functionalized library was incubated first with the HEK293 and followed by the incubation with GluR1-HEK293 cells for 30 min at 37°C and 5% CO2. Afterward, the supernatant was discard and the cells were washed with DMEM.
Finally, the cells were scraped in 500 µL ddH2O and transferred to a 2 mL tube. The bound sequences were recovered by heating for 5 min to 95°C followed by phenol/chloroform extraction (section 8.1.3.2). The purified DNA was used as the template in the PCR. The detailed selection conditions are summarized in Table 8.13 and Table 8.14.
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Table 8.13 Summary conditions of click-SELEX targeting GluR1-HEK293 using indole azides (1).
SELEX
cycle clicked-library HEK293 cells
in T75 flask
GluR1-HEK293 cells
in T25 flask Wash at RT
1 500 pmol - 2.5 ∙ 106 2x 30 sec
2 70 pmol - 2.5 ∙ 106 2x 60 sec
3 30 pmol 5 ∙ 106 2.5 ∙ 106 2x 60 sec
4 30 pmol 3x 5∙ 106 2.5 ∙ 106 2x 60 sec
Table 8.14 Summary conditions of click-SELEX targeting GluR1-HEK293 using benzyl azide (2) and guanidine (11).
SELEX
cycle clicked-library HEK293 cells
in T75 flask
GluR1-HEK293 cells
in T25 flask Wash at RT
1 500 pmol 5 ∙ 106 2.5 ∙ 106 2x 30 sec
2 60 pmol 5 ∙ 106 2.5 ∙ 106 2x 60 sec
3 30 pmol 2x 5 ∙ 106 2.5 ∙ 106 2x 60 sec
4 30 pmol 2x 5 ∙ 106 2.5 ∙ 106 2x 60 sec
5 30 pmol 3x 5 ∙ 106 2.5 ∙ 106 2x 60 sec