DNA-グラフェン酸化物の相互作用におけるDNA構造の影響

1. Biomolecular design laboratory
11491014 Yusuke Sakashita
Effect of DNA structure on
graphene oxide–DNA
interaction

2. Property of graphene oxide (GO)
Ｐroperties of GO
○quenching fluorescence due to large aromatic surface
○Water soluble due to functional groups introduced on GO
○Interaction with biomolecules
Graphene
Oxidization
Graphene oxide (GO)
GO is promising material for biological applications.
R. Li et al., Int. J. Mol. Sci., 14, 12863-12872 (2013).
M. Wu et al., Langmuir, 27, 2731–2738 (2011).

3. Z. Zhang et al., Analyst, 139, 4805-4809 (2014).
H. Volvusha et al., J. Phys. Chem. Lett, 4, 3710−3718 (2013).
Biosencer based on GO for detection of DNA
Adsorption
Probe DNA
with FAM
Desorption
Target DNA
1. π-π stacking
2. Hydrogen bonding
GO-DNA interaction is useful to develop biosensors.
Fluorescence
quenching
Fluorescence
recovery

4. Biosencer based on GO for detection of DNA
GO-DNA interaction is useful to develop biosensors.
Z. Zhang et al., Analyst, 139, 4805-4809 (2014).
H. Volvusha et al., J. Phys. Chem. Lett, 4, 3710−3718 (2013).
Adsorption
ssDNA with FAM
Desorption
Target DNA
1. π-π stacking
2. Hydrogen bonding
Fluorescence
quenching
Fluorescence
recovery

5. Mechanism of interaction between GO and DNA
The mechanism of GO–DNA interaction has been
elucidated for single strand DNA.
The interaction between DNA and GO was performed
by some mechanisms.
T. Paul et al., J. Phys. Chem. B, 20(45),11628-11636 (2016).

6. Problem
How is the secondary structure involved in the interaction
between GO and DNA?
Base paired
region
Unpaired
region
Hairpin loop
DNA
G-quadruplex
DNA
Single strand
DNA
Hoogsteen
Base pair

7. Problem
How is the secondary structure involved in the interaction
between GO and DNA?
Base paired
region
Unpaired
region
Hairpin loop
DNA
G-quadruplex
DNASingle strand
DNA
Hoogsteen
Base pair

8. Materials and methods
Probe DNA : ssDNA (random coil)
5’- [FAM] - GATCCTACTGCTTTCTCTATCG -3’
hpDNA (hairpin loop)
5’- [FAM] - TTCTCTTCTTTTTAGAAGAGAA -3’
gqDNA (G-quadruplex)
5’- [FAM] - AGGGTTAGGGTTAGGGTTAGGG -3’
Target DNA : T-ssDNA (random coil)
5’- ACT GGG GGA GGC GCA AGG -3’
T-hpDNA (hairpin loop)
5’- TTC TCT TTT TAG AGA A -3’
Molecular weight of GO unit
Τ𝟕𝟐. 𝟎𝟔 𝟐 = 𝟑𝟔. 𝟎𝟑
ssDNA hpDNA gqDNA

9. Table. Dissociation constant (𝐾 𝑑) of the probeDNAs with
GO at 24℃ in 100 mM NaCl and 50 mM Tris buffer.
𝑭 = −∆𝑭
𝑮𝑶 𝒏
𝑲 𝒅
𝒏
+ 𝑮𝑶 𝒏
+ 𝑭𝒊𝒏𝒕
∆𝑭 : Final fluorescence intensity change
𝑭𝒊𝒏𝒕 : Initial fluorescence intensity
𝑲 𝒅 : Dissociation constant
n : Hill constant
0
5000
10000
15000
20000
503 511 519 527 535 543 551 559
probe-DNAs
Fluorescenceintencity
GO (mM)
0 mM GO
1 mM
Figure. Fluorecsence spectra of the 5 nM FAM-labeled
ssDNA with 0 – 1 mM GO in 100 mM NaCl and 50 mM Tris
buffer (left).
Effects of the structure of probe DNA
on the GO–DNA adsorption
Kd (mM) n
ssDNA 0.57 ± 0.005 2.2 ± 0.4
hpDNA 0.13 ± 0.008 2.6 ± 0.4
gqDNA 0.091 ± 0.018 1.5 ± 0.5
Secondary structure does not affext the GO-DNA adsorption.
⇒ DNAs may not form secondary structure on GO surface.

10. Effects of salt concentration
on the GO–DNA adsorption
Figure. Fluorescence intensity of 5 nM probe-ssDNA
(left), probe-hpDNA (right) and 0.001 -1 mM GO in a
0(yellow), 10(red), 30(blue), 50(green), 80(violet),
100(orange) mM NaCl and 50 mM Tris buffer (pH 7.5).
ssDNA hpDNA
NaCl
(mM)
Kd
(mM)
n Kd
(mM)
n
0 0.13±0.02 2.1±0.6 0.12±0.02 2.1±0.6
10 0.10±0.02 2.4±0.7 0.27±0.04 3.6±1.3
30 0.072±0.015 1.5±0.4 0.17±0.02 2.6±0.5
50 0.072±0.008 1.8±0.4 0.13±0.007 3.0±0.4
80 0.015±0.004 0.82±0.16 0.11±0.01 1.8±0.4
100 0.056±0.35 2.1±0.4 0.13±0.008 2.6±0.4
F.I.at520nm
Salt concentration does not affect the GO-DNA adsorption.
⇒ Electrostatic repulsion between DNA and GO
does not inhibit the GO-DNA adsorption.
F.I.at520nm
Table 2. Dissociation constnt (Kd) and hill coeffient of
the GO-DNA adsorption with various salt concentration

11. 0
1000
2000
3000
4000
5000
6000
0 200 400 600 800 1000
probe-DNAs and target-DNAs
F.I.at520nm
target-DNA (nM)
ssnc
ƒGƒ‰•[’l
320.61301.17m1
0.221630.68979m2
22756538.6m3
193.18319.93m4
NA2.1554e+5ƒJƒC‚Q•æ
NA0.99497R
sshp
ƒGƒ‰•[’l
891.09397.47m1
0.203010.49785m2
1580.22816.2m3
109.75300.05m4
NA53499ƒJƒC‚Q•æ
NA0.99061R
hpnc
ƒGƒ‰•[’l
143.08207.86m1
0.170950.69479m2
1240.95560.6m3
146.94242.11m4
NA1.1948e+5ƒJƒC‚Q•æ
NA0.9967R
hphp
ƒGƒ‰•[’l
1696.9719.72m1
0.241360.57993m2
2390.93368.9m3
99.086282.68m4
NA50597ƒJƒC‚Q•æ
NA0.99247R
gqnc
ƒGƒ‰•[’l
6.070270.98m1
0.114391.1058m2
151.433884.5m3
56.612650.72m4
NA24152ƒJƒC‚Q•æ
NA0.99928R
gqhp
ƒGƒ‰•[’l
373.26118.73m1
0.682620.60882m2
19622174.8m3
342.24359.33m4
NA5.4954e+5ƒJƒC‚Q•æ
NA0.91777R
Figure. Fluorescence intensity at 520 nm of ssDNA and
T-ssDNA(yellow),ssDNA and T-hpDNA(red), hpDNA and
T-ssDNA(sky blue), hpDNA and T-hpDNA(green), gqDNA
and T-ssDNA(orange) and gqDNA and T-hpDNA(violet) in
100 mM NaCl and 50 mM Tris buffer.
𝑭 = −∆𝑭
𝑫𝑵𝑨 𝒏
𝑲 𝟎.𝟓
𝒏
+ 𝑫𝑵𝑨 𝒏
+ 𝑭𝒊𝒏𝒕
Effects of the structure of target DNAs
on the GO–DNA desorption
Probe DNA Target DNA K0.5 (nM) n
ssDNA T-ssDNA 3.0×102 0.69
T-hpDNA 4.0×102 0.50
hpDNA
T-ssDNA 2.1×102 0.70
T-hpDNA 7.2×102 0.58
gqDNA
T-ssDNA 7.1×102 1.1
T-hpDNA 1.2×102 0.61
●ssDNA + T-ssDNA
●hpDNA + T-ssDNA
●gqDAN + T-ssDNA
●ssDNA + T-hpDNA
●hpDNA + T-hpDNA
●gqDNA + T-hpDNA
DNA desorption from GO depends on
secondary structure of target DNA.

12. Discussion and conclusions
The DNA–GO adsorption does not depend on probe DNA structure
and salt concentration.
DNA may be denatured when it adsorbs onto GO surface.
Secondary structure of the target DNA largely affect the its desorption
from GO.
Sensitivity of target DNA detection may be controlled by its
structure.
ssDNA hpDNA gqDNA
It is necessary to elucidate the adsorption / desorption mechanisms of
DNA forming secondary structures. More systematic and kinetic
studies will be performed.
Future works