M-1NH2. 3. 7.1. 6.9. 4.9. 2.7. 2.3. 2.0. 10. 3.1. 6.9. −1.8. 0.6. 0.4. 2.6. 0. 11.3. 9.1. 13.7. 5.7. 5.5. 0.4. M-1PY. 3. 10.5. 8.6. 9.0. 3.9. 4.7. 1.7. 10. 7.9. 9.3. 7.7. 4.7. 4.0.
SUPPLEMENTARY INFORMATION
Probing of G-Quadruplex Structures via Ligand-Sensitized Photochemical Reactions in BrU-Substituted DNA Abhijit Saha,1,2 Sophie Bombard,1,2 Anton Granzhan,*,1,2 & Marie-Paule Teulade-Fichou*,1,2
1
CNRS UMR9187, INSERM U1196, Institut Curie, PSL Research University, 91450 Orsay, France
2
CNRS UMR9187, INSERM U1196, Université Paris Sud, Université Paris Saclay, 91450 Orsay,
France
Table S1. Ligand-induced stabilization (∆T½ / °C) of G4-DNA from FRET-melting experiments. a Ligand
PY-NH2
M-1NH2
M-1PY
M-2PY
PhenDC3
T½ / a
°C g
cds26 / µMb
G4-DNA (F-G4-T) 22AG (K+) c 22AG (Na+)d
21CTA c
Pu24T e
CEB25WT e
CEB25L111 e,f
0
7.6
9.5
4.8
2.2
3.7
0.7
3
8.5
3.5
4.8
0.3
3.2
2.1
10
7.1
5.1
−0.9
1.1
1.9
2.6
0
15.6
8.6
10.8
3.7
7.3
−0.4
3
7.1
6.9
4.9
2.7
2.3
2.0
10
3.1
6.9
−1.8
0.6
0.4
2.6
0
11.3
9.1
13.7
5.7
5.5
0.4
3
10.5
8.6
9.0
3.9
4.7
1.7
10
7.9
9.3
7.7
4.7
4.0
2.2
0
28.3
22.9
21.7
17.5
24.5
11.0
3
24.1
19.5
19.1
13.6
16.5
10.0
10
24.1
20.0
15.4
10.0
17.5
9.2
0
30.8
23.3
29.3
30.2
31.9
13.2
3
32.6
23.4
28.9
28.4
31.0
16.8
10
32.8
18.7
28.5
24.6
25.8
15.0
0
57.5
57.7
61.9
56.2
54.4
68.5
Conditions: cF-G4-T = 0.2 µM, cligand = 1 µM (except for CEB25L111). c
d
b
Concentration of the double-
stranded competitor (ds26: Table 1). In K-10 buffer. In Na-10 buffer. In K-1 buffer. f cF-G4-T = 0.2 µM, cligand = 0.2 µM. g Melting temperature in the absence of ligands.
e
b) 22AG (Na+)
0.8
0.8
0.8
0.6
F / F0
1.0
0.6
0.4
0.4
0.2
0.2
0.2
2
4
6
8
10
0
2
c(DNA) / µM
4
6
8
10
0
e) hras-1
0.8
0.8
F / F0
0.8
F / F0
1.0
0.6
0.4
0.4
0.2
0.2
0.2
4
6
8
0
10
2
4
6
8
10
12
0
g) CEB25L111
h) Pu24T
0.8
0.8
F / F0
0.8
F / F0
1.0
0.6
0.4
0.4
0.2
0.2
0.2
4
6
8
c(DNA) / µM
10
12
0
2
6
8
10
0.6
0.4
2
4
i) ds26
1.0
0.6
10
c(DNA) / µM
1.0
0
2
c(DNA) / µM
c(DNA) / µM
8
0.6
0.4
2
6
f) CEB25WT
1.0
0.6
4
c(DNA) / µM
1.0
0
2
c(DNA) / µM
d) 22CTA
F / F0
0.6
0.4
0
F / F0
c) bcl2Mid
1.0
F / F0
F / F0
a) 22AG (K+) 1.0
4
6
8
c(DNA) / µM
10
12
0
2
4
6
8
10
12
c(DNA) / µM
Figure S1. Binding isotherms from spectrofluorimetric titrations, presented as a relative change of the emission intensity of the excimer band of M-2PY (F/F0, at λem = 474 nm) in the presence of various DNA substrates, as indicated in each panel. Red lines represent the fitting to the independent-site model using a 2:1 stoichiometry and binding constant (Ka) values given in Table 2. Conditions: c(M-2PY) = 2 µM in K-100 buffer (except for Na-100 buffer in panel b), excitation wavelength: 347 nm. .
S2
a) 22AG (K+) 100
30 20 10
100
[] / 106 (deg cm2 dmol-1)
[] / 106 (deg cm2 dmol-1)
[] / 106 (deg cm2 dmol-1)
c) bcl2MidT20
b) bcl2Mid
40
50
0
50
0
0 -50
-10 240
280
300
240
320
d) 22AG (Na+)
260
280
300
-50
320
240
60
e) 22CTA
260
280
300
320
260
280
300
320
260
280
300
320
f) hras-1
20 0 -20 -40
[] / 106 (deg cm2 dmol-1)
100
[] / 106 (deg cm2 dmol-1)
[] / 106 (deg cm2 dmol-1)
40
260
50
0
40 20 0 -20
-60
-40 -80
-50 240
260
280
300
320
240
300
g) CEB25WT
260
280
300
320
240
300
h) CEB25L111
i) Pu24T
200
100
0
200
[] / 106 (deg cm2 dmol-1)
[] / 106 (deg cm2 dmol-1)
[] / 106 (deg cm2 dmol-1)
300
100
0
200
100
0
-100
-100
-100
-200
240
260
280
300
320
240
260
280
Wavelength (nm)
400
j) Pu24TT21
300
320
240
Wavelength (nm)
T U
Br
[] / 106 (deg cm2 dmol-1)
300 200 100 0 -100 -200 240
260
280
300
320
Wavelength (nm)
Figure S2. CD spectra of unmodified (black) and BrU-modified (red) G4-DNA structures (c = 10 µM in K-100 buffer, except for 22AG (Na+): Na-100 buffer). The sequences are provided in Table 1.
S3
M-1PY
M-2PY
UV (min)
0 3 3 5 0 3 3 5
Ligand
– – + + – – + +
Figure S3. PAGE analysis of photo-irradiated reaction mixtures of unmodified 22AG with M-1PY and M-2PY in K+ conditions. Conditions: c(DNA) = 5 µM, c(ligand) = 25 µM, irradiation with 365 nm UV light (300 W, 3 and 5 min).
S4
Figure S4. Reaction cascades corresponding to a) pyrene-sensitized photoclevage of
BrU-modified
DNA sequence (top row: no additives, bottom
row: i-PrOH / UDG treatment) and b) DMS piperidine induced cleavage on BrU-modified G4-DNA. A fragment of the human telomeric repeat sequence (5′-…AGGGBrUBrUAGG…-3′) is shown in both cases, highlighting the difference in cleavage sites.
PY-NH2 – + + + UV (min) 0 0 3 3
** *
Figure S5. PAGE analysis of BrU-22AG irradiated in the presence of PY-NH2 (10 molar equiv.) in K+ conditions. Conditions: c(BrU-22AG) = 5 µM in K-100 buffer, irradiation with 365 nm UV light (300 W). Band assignment: *, unmodified DNA band; **, presumable covalent adduct. a) Ligand (eq.) UV (min)
M-1PY
M-2PY
0 1 2 5 10 20 5 5 5 5 5 5
b)
M-1PY
M-2PY
0 1 2 5 10 20
5 5 5 5 5 5
5 5 5 5 5 5
5 5 5 5 5 5
0 1 5 10 20 30
0 1 5 10 20 30
** *
Figure S6. PAGE analysis of BrU-22AG irradiated in the presence of ligands M-1PY and M-2PY: a) using varied concentrations of the ligands; b) using different irradiation times, as indicated in the caption. Conditions: c(BrU-22AG) = 5 µM in K-100 buffer, irradiation with 365 nm UV light (300 W).
M-1PY M-2PY 20
b)
Photoadduct yield (%)
Photoadduct yield (%)
a)
10
M-1PY M-2PY
20
10
0
0 0
5
10
15
0
20
10
20
30
Irradiation time (min)
Ligand-to-DNA ratio (equiv.)
Figure S7. Influence of a) ligand-to-DNA ratio (using fixed irradiation time: 5 min, 365 nm) and b) irradiation time (using fixed ligand-to-DNA ratio: 5 molar equivalents) on the yield of the photoadducts (**) formed in ligand-sensitized photoreactions with
BrU-22AG.
Conditions as indicated for Figure S4.
Data from densitometric analysis of gel electrophoresis data; error bars represent standard deviation from two independent experiments.
S7
DNA
a) M-1PY (K+)
b) M-1PY (Na+)
22AGL1 22AGL2 22AGL3
22AGL1 22AGL2 22AGL3
UV (min) 3 3 5 3 3 5 3 3 5 Ligand − + + − + + − + + Lane 1 2 3 4 5 6 7 8 9
c) M-2PY (K+)
3 3 5 3 3 5 3 3 5 − + + − + + − + + 10 11 12 13 14 15 16 17 18
d) M-2PY (Na+)
DNA
22AGL1 22AGL2 22AGL3
22AGL1
22AGL2 22AGL3
Lane
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18
e)
22AGL1: 5′-AGGG-BrUBrUA-GGG-TTA-GGG-TTA-GGG 22AGL2: 5′-AGGG-TTA-GGG-BrUBrUA-GGG-TTA-GGG 22AGL3: 5′-AGGG-TTA-GGG-TTA-GGG-BrUBr UA-GGG
Figure S8. PAGE analysis of photoreactivity of each loop of 22AG towards M-1PY (a–b) and M-2PY (b–c), assessed by variable substitution by BrU, both in K+ (a, c) and in Na+ (b, d) conditions. Conditions: c(DNA) = 5 µM, c(ligand) = 25 µM, irradiation with 365 nm UV light (300 W). Lanes 1, 4, 7, 10, 13, 16: UV-irradiated DNA controls, lanes 2, 3, 11 and 12: 22AG L1 irradiated in the presence of ligands, lanes 5, 6, 14, 15: same for 22AGL2, lanes 8, 9, 17, 18: same for 22AGL3. For the sequences of 22AGL1, 22AGL2 and 22AGL3, cf. Figure 4 in the main text.
S8
a) M-1PY UV (min) Ligand ct DNA (mM)
0 0 3 3 – + + + 0 0 0 0
3 3 3 3 + + + + 2 2 4 4
3 3 + + 6 6
b) M-2PY 3 3 + + 8 8
3 3 + + 10 10
3 3 3 3 3 3 + + + + + + 12 12 16 16 20 20
0 0 – + 0 0
3 + 0
3 + 0
3 + 2
3 + 2
3 + 4
3 + 4
3 + 6
3 + 6
** *
3 + 8
3 3 3 3 3 3 3 + + + + + + + 8 10 10 16 16 20 20
** *
Figure S9. PAGE analysis of photoreaction of
BrU-22AG
(5 µM in K-100 buffer) with a) M-1PY and b)
M-2PY (25 µM in each case) in the presence of increasing amounts of ct DNA competitor, as indicated in the header (0 to 20 mM). In panel b), both parts of the images are parts of the same gel. Quantification of these gels is shown in Figure 7 of the main text.
22AG2
Br
U-22AG
UV (min)
−
− 3 3 5
M-2PY
−
− − + +
** *
Figure S10. PAGE analysis of the products of a photoreaction of
BrU-22AG
with M-2PY and the
comparison with a 44-nt oligonucleotide fragment [22AG2: 5′-A(GGGTTA)3GGGA(GGGTTA)3GGG-3′]. *: Unmodified DNA band, **: presumable covalent adduct.
S9
Lane
a) *
b) **
0 1 2 3
0123
**
*
arrest
Figure S11. PAGE analysis of snake venom exonuclease treatment of unreacted BrU-22AG (*, panel a) and the putative covalent crosslinked product, formed in the photoreaction of BrU-22AG with M-2PY and excised from electrophoresis gel (**, panel b). Lane 0 in both panels: no exonuclease treatment, lane 1: treatment with 0.04 U, lane 2: 0.02 U, lane 3: 0.01 U of snake venom exonuclease in Tris-HCl, 5 mM MgCl2 (pH 7.5) in the presence 0.5 mg mL−1 tRNA; incubation at 37 °C for 30 min.
S10
0
1
2
2′
1′
0′
** *
Figure S12. Full gel corresponding to Figure 9, a (with alignment marks). PAGE analysis of the products of a photoreaction of non-labeled BrU-22AG (50 µM) with M-2PY (125 µM). Top panel: UV shadowing image of the electrophoresis gel, obtained under UV light (λ = 254 nm) with the help of a TLC plate with F254 indicator. Bottom panel: auto-fluorescence image of the gel with UV transillumination (G:Box, λ = 305 nm). Lanes 0 and 0′: reference dye (Xylene Cyanol FF), lanes 1 and 1′: non-irradiated control, lanes 2 and 2′: photo-irradiated mixture (365 nm, 3 min). *: Unmodified substrate band; **: covalent adduct band; ○: Xylene Cyanol FF; +: reference marks used for image alignment.
S11
b) a)
Fluorescence intensity (a.u.)
1.5x104
* **
irradiated sample
1.0x104
non-irradiated sample
** * 5.0x103
c) 0.0 350
400
450
500
**
Wavelength (nm)
*
Figure S13. Fluorescence spectroscopy analysis of the photoproduct of reaction of
BrU-22AG
(50 µM)
with M-2PY (125 µM). a) Background-corrected fluorescence emission spectra (λ ex = 345 nm) of DNA fragments extracted from the electrophoresis gel: * control DNA band from the non-irradiated reaction mixture; ** slow-migrating band corresponding to the putative covalent ligand–DNA adduct. b–c) UV shadowing image of the electrophoresis gel, b) before and c) after cutting of the indicated bands (* and **). The image was obtained under UV light (λex = 254 nm) using a TLC plate with an F254 indicator. The bands were cut, soaked in 500 µL of 0.15 M NaCl solution at 37 °C overnight, and the supernatant was analyzed by fluorescence spectroscopy.
S12
AS180418-PyNH2-BrU_0min
2: Diode Array 254 Range: 7.819e-1
3.56
a)
BrdU
6.0e-1
AU
5.02
PY-1NH2
4.0e-1 2.0e-1
6.33
0.0 1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00 2: Diode Array 350 Range: 3.19e-1
7.00
8.00
9.00
10.00 2: Diode Array 254 Range: 9.111e-1
7.00
8.00
9.00
10.00 2: Diode Array 350 Range: 3.495e-1
8.00
9.00
10.00 1: Scan ES+ 630 4.44e6
AS180418-PyNH2-BrU_0min 5.02
3.0e-1
b)
PY-1NH2
AU
2.0e-1
1.0e-1 6.33
0.0 1.00
2.00
3.00
4.00
5.00
6.00
AS190418PyNH2_BrU_3h30 3.57
c)
BrdU
AU
6.0e-1
5.00
PY-1NH2
4.0e-1 2.0e-1
6.31
1.57
4.55
0.0 1.00
2.00
3.00
4.00
5.00
6.00
AS190418PyNH2_BrU_3h30 5.00
AU
3.0e-1
d)
PY-1NH2
2.0e-1
1.0e-1 4.13
6.31
4.45 4.55
5.30 5.55
6.77
0.0 1.00
2.00
3.00
4.00
5.00
6.00
7.00
6.00
7.00
AS190418PyNH2_BrU_3h30 100
4.54
e)
4.44 4.59
%
4.12
4.83
5.39
7.70
8.17 8.36
0 1.00
2.00
3.00
4.00
5.00
8.00
9.00
Time 10.00
Figure S14. LC/MS chromatograms of a mixture of PY-1NH2 (1 mM) and BrdU (10 mM in 10 mM LiAsO2Me2 buffer, pH 7.2), before (a–b) and after (c–d) UV irradiation (365 nm, 3 h 30 min). Panels a) and c): single-wavelength chromatograms (λ = 254 nm); panels b) and d): single-wavelength chromatograms (λ = 350 nm); panel e): single-ion chromatogram (m/z = 630) corresponding to the expected covalent adducts. The red arrows indicate the putative covalent adduct peaks.
S13
AS190418PyNH2_BrU_3h30 331 (5.015) Cm (326:336) 100
1: Scan ES+ 1.56e7
403.4
a) PY-1NH2 [M + H]+
257.2
193.9
%
202.3
404.4
300.3
185.3 215.2 150.7
258.3
130.2 301.3
274.2 216.2
343.3
0 150 200 250 300 350 AS190418PyNH2_BrU_3h30 300 (4.545) Cm (271:305)
400
450
500
550
600
650
700
750
800
850
900
950
800
850
900
950
257.3
100
m/z 1000 1: Scan ES+ 5.02e5
b)
[PY-1NH2 + BrdU – Br–]+ 248.8
%
629.5
171.1
513.5
117.0 269.3
240.4
367.3
410.2
630.6
384.3 155.1 172.1
514.4 411.2
227.3
631.5
312.3
453.4
470.4
741.5
0 150
200
250
300
350
400
450
500
550
600
650
700
750
m/z 1000
Figure S15. Mass spectra of peaks eluted at a) tR = 5.0 min (PY-1NH2, M = 402.6 g/mol) and b) 4.1– 4.5 min (putative covalent adduct with BrdU, M = 628.8 g/mol).
S14
