Supporting Information
Wiley-VCH 2013
69451 Weinheim, Germany
Combination of i-Motif and G-Quadruplex Structures within the Same
Strand: Formation and Application**
Jun Zhou, Samir Amrane, Dursun Nizam Korkut, Anne Bourdoncle, Hong-Zhang He,
Dik-Lung Ma, and Jean-Louis Mergny*
ange_201301278_sm_miscellaneous_information.pdf
Sequence Information
As described in main text, the italicized and underlined bases in MIX should form
i-motif and G-quadruplex structures, respectively. “Mutations” are shown in red for
the 6 control sequences:
Table S1: Sequence of all tested oligonucleotides:
5’
CCCCTTCCCCTGGGTGGGTTGGGTGGGTCCCCTTCCCC3’
5’
CCCCTTCCCCTTTTTTTTTTTTTTTTTTCCCCTTCCCC3’
5’
TTTTTTTTTTTGGGTGGGTTGGGTGGGTTTTTTTTTTT3’
5’
CCCCTTCTTCTGGGTGGGTTGGGTGGGTCCCCTTCCCC3’
5’
CCCCTTCCCCTGGGTTTGTTGGGTGGGTCCCCTTCCCC3’
5’
CCTCTTCTTCTGGGTGGGTTGGGTGGGTCTCCTTCTTC3’
5’
CCCCTTCCCCTGGTTGTGTTGTGTTGGTCCCCTTCCCC3’
(MIX)
(ConC)
(ConG)
(MutC)
(MutG)
(MutC2)
(MutG2)
Materials and Methods
Oligonucleotides listed above were purchased from Eurogentec (Seraing) or
Integrated DNA Technologies (Leuven) without further purification (Reverse-Phase
Cartridge Gold, desalting or PAGE purified). Concentrations were determined by
ultraviolet (UV) absorption using the extinction coefficients provided by the
manufacturer. All chemicals used were purchased from Sigma.
Sample preparation
The samples were heated to 95 oC for 5 min, cooled on ice quickly, and then stored at
4 oC more than 6 hours in 10 mM lithium cacodylate (pH 5.8 or 7.4) in the presence or
absence of 50 mM KCl before use unless otherwise stated. The concentration of
oligos is 4 µM. Therefore, except when stated otherwise, conditions labelled as "pH
5.8, 50 mM K+" actually correspond to 10 mM lithium cacodylate buffer adjusted at
pH 5.8 and supplemented with 50 mM KCl.
For NMR experiments, the buffer was 20 mM MES (pH 5.8) or 20 mM HEPES (pH
7.4) in the presence or absence of 50 mM K+, and the concentration of oligos is 100
µM.
UV-melting curves,
measurements
thermal
difference
spectra
and
time-dependent
UV-absorbance measurements were performed with Uvikon XL or XS (Secomam)
equipped with a circulating water bath. Temperature was measured with an inert glass
sensor immersed into a quartz cell.
- For melting experiments, the absorbance was monitored at 295 nm with a
heating rate of ~0.19 oC/min. For each sample, the thermal difference spectrum (TDS)
was obtained by subtracting the absorbance spectrum at low temperature (around 20
o
C) from the one at around 60 oC or that at around 60 oC from the one at high
temperature (around 90 oC). For comparison, TDSs of the absorbance spectrum at low
minus high temperature were also plotted.
- For time-dependent measurements, absorbance was set at 295 nm, and
concentrated HCl, LiOH or KCl was added and rapidly mixed with the sample. The
temperature was set at 25 oC and the dead time is about 1 min.
Circular dichroism (CD) spectroscopy
Circular dichroism spectra were recorded on a JASCO J-815 equipped with a Peltier
temperature control accessory. Each spectrum was obtained by averaging five scans at
a speed of 100 nm/min. A background CD spectrum of corresponding buffer solution
was subtracted from the average scan for each sample. CD melting curves were
obtained by measuring the CD spectra of samples from low temperature to high
temperature with an average temperature increasing rate of ~1 oC/min. The data at
260 nm or 286 nm were plotted against temperature.
Fluorescence spectroscopy
Fluorescence spectra were recorded on a Fluoromax-4 sepctrofluorometer (HORIBA
Scientific) at room temperature (temperature was controlled by circulating water bath,
which was fixed at 25 oC). Each spectrum was obtained by averaging five scans. The
concentration of oligonucleotide was 4µM, and that of CV was 1µM.
Figure S1
Figure S1. Chemical structure of crystal violet (CV).
Figure S2
a)
50
b)
40
40
20
10
0
C onC pH 5.8
C onC pH 5.8, 50 m M K
C onG pH 5.8, 50 m M K
C onG pH 7.4, 50 m M K
-­‐10
-­‐20
220
240
260
280
300
320
C D / m d e g
C D / m d e g
30
-­‐30
50
30
20
10
0
MutC , pH 5.8, 50 m M K MutC , pH 7.4, 50 m M K MutG , pH 5.8
MutG , pH 5.8, 50 m M K -­‐10
-­‐20
340
220
240
λ / n m
260
280
300
320
340
λ / n m
c ) 50
40
C D / m d e g
30
20
10
0
MutC 2, pH 5.8, 50 m M K
MutC 2, pH 7.4, 50 m M K
MutG 2, pH 5.8, 50 m M K
MutG 2, pH 7.4, 50 m M K
-­‐10
-­‐20
-­‐30
220
240
260
280
300
320
340
λ / n m
Figure S2. CD spectra of control sequences a) ConC and ConG; b) MutC and MutG;
and c) MutC2 and MutG2 pH 5.8 or 7.4 in the presence or absence of 50 mM K+. The
data were collected at 20 oC.
Figure S3
b)
a)
0.32
C onC pH 5 .8
C onC pH 5 .8 , 5 0 m M K
C onG pH 5 .8 , 5 0 m M K
C onG pH 7 .4 , 5 0 m M K
0.30
0.36
0.34
0.32
A 2 9 5 n m
0.28
A 2 9 5 n m
MutG , pH 5.8
MutG , pH 5.8, 50 m M K
MutC , pH 5.8, 50 m M K
MutC , pH 7.4, 50 m M K
0.38
0.26
0.24
0.30
0.28
0.26
0.22
0.24
0.20
0.18
0.22
20
30
40
50
60
70
80
0.20
90
20
o
T / C
40
50
60
70
80
90
o
T / C
c)
MutC 2, pH 5.8, 50 m M K
MutC 2, pH 7.4, 50 m M K
MutG 2, pH 5.8, 50 m M K
MutG 2, pH 7.4, 50 m M K
0.32
0.28
A 2 9 5 n m
30
0.24
0.20
0.16
0.12
0.08
20
30
40
50
60
70
80
90
o
T / C
Figure S3. UV melting profiles of control sequences a) ConC and ConG; b) MutC
and MutG; and c) MutC2 and MutG2 under various buffer conditions.
Figure S4
C D a t 260 n m o r 286 n m
b)
pH 5.8, 286 nm
pH 5.8, 260 nm
pH 5.8, 50 m M K ,286 nm
pH 5.8, 50 m M K ,260 nm
MIX
50
40
30
20
10
40
30
20
10
20
0
20
30
40
50
60
70
80
pH 7.4, 286 nm
pH 7.4, 260 nm
pH 7.4, 50 m M K , 286 nm
pH 7.4, 50 m M K , 260 nm
MIX
50
C D a t 260 n m o r 286 n m
a)
30
40
50
60
70
80
90
o
T / C
90
o
T / C
C D a t 260 n m o r 286 n m
c)
C onG ,pH 5.8, 50 m M K , 260 nm
C onG ,pH 7.4, 50 m M K , 260 nm
C onC ,pH 5.8, 286 nm
C onC ,pH 5.8, 50 m M K , 286 nm
40
30
20
10
C o n C & C o n G
0
20
40
60
80
o
T / C
50
Mu tC e)
pH 5.8, 50 m M K , 286 nm
pH 5.8, 50 m M K , 260 nm
pH 7.4, 50 m M K , 286 nm
pH 7.4, 50 m M K , 260 nm
40
C D a t 260 n m o r 286 n m
C D a t 260 n m o r 286 n m
d)
30
20
10
0
20
30
40
50
60
70
80
pH 5.8, 286 nm
pH 5.8, 260 nm
pH 5.8, 50 m M K , 286 nm
pH 5.8, 50 m M K , 260 nm
40
30
20
10
0
-­‐10
90
Mu tG 50
o
20
30
40
T / C
C D a t 260 n m o r 286 n m
pH 5.8, 50 m M K , 286 nm
pH 5.8, 50 m M K , 260 nm
pH 7.4, 50 m M K , 286 nm
pH 7.4, 50 m M K , 260 nm
Mu tC 2 30
20
10
C D a t 260 n m o r 286 n m
g)
50
20
30
40
70
80
90
50
60
o
T / C
70
80
90
pH 5.8, 50 m M K , 286 nm
pH 5.8, 50 m M K , 260 nm
pH 7.4, 50 m M K , 286 nm
pH 7.4, 50 m M K ,, 260 nm
Mu tG 2 40
30
20
10
0
-­‐10
-­‐20
0
60
o
T / C
f)
40
50
20
30
40
50
60
70
80
90
o
T / C
Figure S4. CD melting profiles of a) and b) MIX and its control sequences c) ConC
and ConG; d) MutC and e) MutG; and f and g) MutC2 and MutG2 at 286 nm or 260
nm under various buffer conditions as indicated in each panel.
At pH 5.8 in the presence of K+, CD melting data at 286 nm exhibits a single sigmoid
transition (50 oC, Figure S4a), similar to MIX at pH 5.8 in the absence of K+ (52 oC,
Figure S4a) and control sequences ConC, MutG, and MutG2 at pH 5.8 with or
without K+ (Figure S4c, e and g). In contrast, the CD melting profile at 260 nm shows
two sigmoid transitions (Figure S4a), in agreement with UV-absorbance melting
results described in main text (Figure 1b, ○). The first transition is attributed to i-motif
melting (Tm 52 oC, similar to result obtained in the absence of K+) and the second to
G-quadruplex melting (Tm 72 oC, similar to that of the ConG, MutC, and MutC2
sequences, Figure S4c, d and f).
Figure S5
80
o
T m / C
75
tran s itio n 1
tran s itio n 2
70
50
45
40
0
10
20
30
40
50
c o n c en tratio n o f MIX / µM
Figure S5. Melting temperatures of MIX determined by UV-absorbance at various
concentrations at pH 5.8 in the presence of 50 mM K+.
Figure S6
Figure S6. 1H NMR of MIX and control sequences MutG, MutC, MutG2, and MutC2
at pH 5.8 in the presence of 50 mM K. All the spectra are collected at 25 oC.
Figure S7
a)
C V -­‐pH 5 .8
C V -­‐pH 5 .8 , 5 0 m M K
C V -­‐pH 7 .4
C V -­‐pH 7 .4 , 5 0 m M K
C V -­‐MIX , pH 5 .8
C V -­‐MIX , pH 5 .8 , 5 0 m M K
C V -­‐MIX , pH 7 .4
C V -­‐MIX , pH 7 .4 , 5 0 m M K
700
600
I / a . u .
500
400
300
200
100
0
580
600
620
640
660
680
700
720
740
λ / n m
b)
C V -­‐pH 5.8
C V -­‐pH 5.8, 50 m M K
C V -­‐pH 7.4
C V -­‐pH 7.4, 50 m M K
C V -­‐MIX , pH 5.8
C V -­‐MIX , pH 5.8, 50 m M K
C V -­‐MIX , pH 7.4
C V -­‐MIX , pH 7.4, 50 m M K
100
90
80
I / a . u .
70
60
50
40
30
20
10
0
580
600
620
640
660
680
700
720
740
λ / n m
Figure S7. a) One example of fluorescence spectra a of crystal violet (CV) in the
absence (hollow symbols) or presence (filled symbols) of MIX under different
conditions; b) Enlargement of Figure S7a above for distinguishable graph about those
samples that intensity are much lower than the sample CV in the presence of MIX at
pH 5.8. Temperature was controlled with an external water bath thermostated at 25
o
C.
Figure S8
C V -­‐MIX ,pH 5 .8
C V -­‐MIX ,pH 5 .8 , 5 0 m M K
C V -­‐MutG ,pH 5 .8
C V -­‐MutG ,pH 5 .8 , 5 0 m M K
C V -­‐MutG 2 ,pH 5 .8
C V -­‐MutG 2 ,pH 5 .8 , 5 0 m M K
C V -­‐C onC ,pH 5 .8
C V -­‐C onC ,pH 5 .8 , 5 0 m M K
700
600
I / a . u .
500
400
300
200
100
0
580
600
620
640
660
680
700
720
740
λ / n m
Figure S8. Fluorescence spectra of crystal violet (CV) at pH 5.8 in the absence or
presence of 50 mM K+ with different DNA sequences. Temperature was controlled
with an external water bath thermostated at 25 oC.
Figure S9
a)
MIX A 295
0.16
50 m M K
p H 5.8
0.14
50 m M K
0.12
p H 7.4
0.10
0
10
20
30
40
50
60
HC l
HC l
T / m in
b ) 0.42
MIX HC l
HC l
HC l
HC l
HC l
HC l
HC l
HC l
0.41
pH5
A 295
0.40
w ater
0.39
p H 9
0.38
L iO H
L iO H
0.37
0
20
40
60
L iO H
80
100
L iO H
120
140
160
L iO H
180
200
L iO H
220
240
L iO H
260
280
L iO H
300
320
L iO H
340
360
380
400
T / m in
Figure S9. UV absorbance of MIX at 295 nm under different stimuli.
- In the upper panel, Potassium is added at t=30 minutes. G4 formation leads to an
increase in absorbance at 295 nm that occurs in less than 60s.
- In the lower panel, pH is adjusted by additions of LiOH or HCl. No buffer was
present to facilitate pH adjustment. Temperature was controlled with an external
water bath thermostated at 25 °C.