Structural stability of the photo-responsive DNA duplexes containing one azobenzene: Via a confined pore

Article abstract of DOI:10.1039/c7cc04599a

Herein, the structural stability of single azobenzene modified DNA duplexes, including the trans form and cis form, has been examined separately based on their distinguishable unzipping kinetics from the mixture by an α-hemolysin nanopore. Furthermore, the accurate isomerization efficiency between the trans and cis form was obtained with single molecule resolution.

Full text of DOI:10.1039/c7cc04599a

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Structural stability of the photo-responsive DNA duplexes
containing one azobenzene via a confined pore
Received 00th January 20xx,
Accepted 00th January 20xx
Fu-Na Meng,‡ Zi-Yuan Li,‡ Yi-Lun Ying,* Shao-Chuang Liu, Junji Zhang and Yi-Tao Long
DOI: 10.1039/x0xx00000x
www.rsc.org/
Here, the structural stability of single azobenzene modified DNA different way is necessary.
duplexes, including the trans form and cis form, has been
The α-hemolysin (α-HL) nanopore,10 a single molecule tool,
examined separately based on their distinguishable unzipping has been proven as an ultra-sensitive system for the
kinetics from the mixture by an α-hemolysin nanopore. Further, determination of individual molecules, such as ssDNA,11,12
the accurate isomerization efficiency between the trans and cis peptides,13,14 proteins15,16 and host-guest molecules.17,18
form was obtained with single molecule resolution.
Previous researches have incorporated α-HL nanopores for
studying the duplex structural stability influenced by a single
base lesion.19,20 In this paper, we employed electrochemical
confined effect of α-HL nanopore to discriminate the trans
form and cis form of azo-dsDNA from the mixtures and explore
their structural stability, respectively (Fig. 1). The azo-dsDNA
was electrochemically driven into the nanopore following by
sequentially unzipping and translocating through the pore. The
confined space in α-HL converts the structural stability of
Photo-responsive DNA modified with azobenzene is an
attractive design for efficiently photo-regulating DNA functions
and DNA nanomachines.1,2 For example, based on the
azobenzene-modified photo-responsive DNA, DNA replication,
transcription and other enzyme activity achieved to be
regulated by artificial operation.3–5 Similarly, DNA
nanomachines, extraordinarily promising in molecular sensing,
logic gate operating and drug delivering, also could be
controlled by light utilizing the photo-responsive DNA, which
avoided the addition of molecular fuels for each working
cycle.6–8 The essential of the photo-controlling DNA is to
regulate the azobenzene-modified DNA duplex (azo-dsDNA)
stability imparted by photo-responsive azobenzene for
ultimate hybridization/dissociation.9 However, the structural
stability of photo-responsive DNA duplex imparted by single
azobenzene was rarely reported. The main reason is that there
exist lots of difficulties in discriminating the trans form and cis
form of azo-dsDNA from the mixtures. For example, although
melting curves9 may provide the structural stability of the
trans form and cis form of azo-dsDNA combining with
calculations, such measurement would be difficult owing to
two reasons. First, an expected Tm change by one azobenzene
could be very small. Second, even if the small variation could
be monitored, the results were derived from the ensemble
solution not reflected the trans form and cis form,
respectively. Therefore, probing the structural stability by
Fig. 1 (a) Schematic illustration of the reversible conversion between the trans and cis
form of azo-dsDNA by photo regulation. A single azobenze moiety is shown in red color.
(b) Discrimination of the trans and cis form of azo-dsDNA from the mixture by an α-HL
nanopore. The α-HL nanopore inserted into a lipid bilayer. The two chambers beside
the nanopore were termed as cap side and stem side, respectively. Individual azo-
dsDNA in the cap side chamber unzips and traverses the α-HL nanopore. The trans form
of azo-dsDNA generated the blockages with longer unzipping time than the cis form,
which showed the high structural stability of the trans form. The voltage was applied
using a pair of Ag/AgCl electrodes. The illustration was not to scale.
Key Laboratory for Advanced Materials & School of Chemistry and Molecular
Engineering, East China University of Science and Technology, Shanghai 200237, P.
R. China.
Email: yilungying@ecust.edu.cn; Fax: +86-21-64250032; Tel: +86-21-64250032
‡
†
These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available. DOI: 10.1039/x0xx00000x.
This journal is © The Royal Society of Chemistry 20xx
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individual azo-dsDNA into distinguishable kinetic unzipping
information. Compared to the native duplex, the trans form
are about 7 times more stable and the cis form around 2 times
less stable. Further, our method achieves to provide an
isomerization efficiency of around 46% (trans-to-cis) and 71%
(cis-to-trans) for the photo-regulated azo-dsDNA based on
single molecule discrimination. The accurate isomerization
efficiency is a vitally important parameter for evaluating the
photo-controlled DNA nanostructures.
The random sequence of azo-ssDNA and its complementary
DNA (c-ssDNA) was as follows:
azo-ssDNA 5’-CGATC-X-CTAGC-3’
c-ssDNA 5’-GCTAG----GATCG-(C)30-3’
One azobenzene group was introduced into position X through
a D-threoninol group (Fig. S1-S14). The azo-dsDNA was formed
with the azobenzene group intercalating between adjacent
base pairs as demonstrated in previous researches2,21. The
Fig. 2 The 2D contour plots (upper) and the duration (t) histograms (bottom) of the
unzipping evens for the annealed DNA solution containing azo-dsDNA after irradiation
by UV (a, b) and vis light (c, d), respectively. Both the 2D plots were composed of 2500
double-stranded segment was followed by
a poly(dC)30
overhang via annealing (see ESI†). The overhang will help the
blockage events. The duration distributions were fitted by
a double exponential
DNA duplex to be captured by α-HL nanopore easily and then
the unzipping events of the duplex can be obtained in
quantity.
function and the fitted parameters were shown in Table S1. Both the histograms were
composed of 1550 events. The experiments were conducted in solutions containing 1.0
M KCl buffered with 10 mM Tris and 1 mM EDTA (pH = 8.0) at +80 mV.
First, to obtain more cis form of the azo-dsDNA, the
annealed solution was irradiated with UV light for 5 min at 58
°C. Then, the solution was added into the cap side chamber at
dark. It produced lots of blockage events at +80 mV. We
extracted the duration times and I/I0 of the events for statistic.
Here I was defined as the blockage residue current and I0 as
the open pore current. In the 2D contour plots, there was one
population distributed in the region with duration less than
0.20 ms (Fig. 2a), which may come from the unbind ssDNA (Fig.
cap side chamber for 10 min. As shown in Fig. 2c-d, the
solution also induced P1 and P2 in both the 2D plot and the
duration histogram for the unzipping events. Compared to the
results under UV irradiation, the event count of P1 decreased
and that of P2 increased evidently in the 2D plot and the
histogram (Fig. 2). These results demonstrated that UV caused
P1 increasing and vis induced P2 rising. To further confirm this
phenomenon, the frequencies for population P1 and P2 were
also analyzed after UV and vis irradiation, respectively (Fig. 3a).
The frequencies of population P1 and P2 under UV irradiation
were 6.0 ± 1.2 and 6.5 ± 0.9 min-1, respectively (Table S2).
When switched to vis light, the frequency of P1 decreased to
2.8 ± 0.5 min-1, while P2 increased to 9.6 ± 1.6 min-1. This
further shows UV light induces P1 increasing and vis gives rise
to P2. It is convinced that UV light could convert the azo-
dsDNA from trans to cis form and on the other hand, vis could
contribute to the trans form.1 Thus, P1 was ascribed to the cis
form and P2 was assigned to the trans form of the azo-dsDNA.
The fitted values of P1 and P2 in the duration histogram were
regarded as unzipping time constants (τ) for the separate cis
and trans form through the α-HL nanopore, which are 0.52 ±
0.01 and 8.07 ± 0.36 ms at +80 mV, respectively. As well as the
duration histograms of the two isomers were both well
described by single exponential function, the rate constants (k)
of the unzipping process were acquired using the following
equation23:
S15, ESI†). Additionally, the collision behavior of the dsDNA
with the nanopore also might induce these short-lived events.
Noticed that there is another clear distribution with long
duration (over 0.2 ms) and deep current amplitude with I/I0
less than 0.23. Since the ssDNA in the solution only induced
short-lived events (Fig. S15, ESI†), the population with duration
over 0.2 ms was ascribed to the azo-dsDNA. According to the
previous research,20 this population was derived from the
duplexes unzipping through the α-HL nanopore (Fig. 1b).
Typically, the unzipping duration of a double-stranded DNA
within the α-HL nanopore is much longer than the
translocation time of the ssDNA at the same applied
potentials.19,22 Therefore, the duration of these current
blockages is deemed as the unzipping times for extracting
kinetic information of strand dissociation related to the duplex
stability. The duration of the azo-dsDNA was fitted into a
double exponential function and two distribution peaks, P1
and P2, were obtained (Fig. 2b). This demonstrate that there
existed two unzipping time constants for azo-dsDNA, which
might be corresponding to the trans and cis form, respectively.
Accordingly, the population in 2D plots was divided into
population P1 and P2 for the following clear statement (Fig.
2a).
k=1/τ
Thus, the rate constants of duplex dissociation for cis form and
trans form were, respectively, 1920 ± 400 s-1 and 124 ± 15 s-1
at +80 mV. The structural stability of the duplexes is
interpreted in terms of their kinetic results.19 Therefore, the
duplex conformation containing trans-azobenzene is more
stable, while the cis-azobenzene instead facilitates the strand
dissociation relative to the native duplex. The trans form with
To further determine the assignment of these two
populations, visible light, which could convert the cis to trans
form of azo-dsDNA, was used to irradiate the solution in the
2 | J. Name., 2012, 00, 1-3
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high stability has to overcome higher energy barrier to unzip
through the nanopore and thus needs more time to endure
the electrical force.
Moreover, the native DNA duplex was analyzed via an α-HL
nanopore to compare with the azo-dsDNA. The native duplex
was formed via the hybridization between the native ssDNA
(5’-CGATCCTAGC-3’) and c-ssDNA. Similarly, lots of unzipping
events were induced by the native duplex (Fig. S15 and S16,
ESI†). In the duration histogram for the unzipping events, the
distribution followed a single exponential function (Fig. 3b),
rather than a double exponential distribution of azo-dsDNA.
UV and vis irradiation hardly affected the histogram
distribution (Fig. S17, ESI†), which further demonstrated that
the double exponential distribution for azo-dsDNA resulted
from the incorporation of azobenzene. The unzipping time
constant for the native duplex was 3.48 ± 0.47 ms at +80 mV.
Thus, the rate constant was 287 ± 32 s-1, which lay between
them for cis form (1920 ± 400 s-1) and trans form (124 ± 15 s-1)
of azo-dsDNA. These results demonstrated that the cis form
has structural instability and trans form high stability relative
to the native duplex. It is suggested that the nonplanar
azobenzene (cis form) may interrupt the base stacking and
hydrogen bonding to destabilize the duplex relative to the
native duplex. While the planar azobenzene (trans form) may
enhance the base stacking to stabilize the duplex.
Fig. 3 (a) Cumulative event count versus recording time (min) for population P1 and P2
in the 2D contour plots under UV or vis irradiation. The curve was fitted linearly and the
fitting parameters were demonstrated in Table S2. (b) Singe exponential distribution of
the duration of unzipping events for the native duplex at +80 mV. The fitting
parameters were exhibited in Table S3. (c) The unzipping time constant for the native
duplex and the cis and trans form of azo-dsDNA as a function of applied voltage. All the
curves were fitted by single exponential function and the fitting parameters were
shown in Table S4. (d) The percentages of cis and trans form of azo-dsDNA under UV
and vis irradiation, respectively. The percentages were obtained via the corresponding
peak area of the duration histogram at +80 mV. The experiments were performed in
solutions containing 1.0 M KCl buffered with 10 mM Tris and 1 mM EDTA (pH = 8.0).
The unzipping events of azo-dsDNA and the native duplex
were also studied at different potentials ranging from +70 to
+110 mV (Fig. 3c and Fig. S18-S19, Table S1 and S3, ESI†). The
traditional method under the similar experiment conditions,
which is about 50% and 85%.25 The little difference of
isomerization efficiency by the two methods not only come
from the slight difference of the target duplexes but also
originate from the sensitivity of these two methods. The
results provided by nanopore technique are much more
precise as they are acquired based on single molecule
discrimination from the heterogeneous mixture. The accurate
isomerization efficiency is an important evaluation index for
the reliable and high-efficiency photo-responsive DNA
nanodevices.
unzipping time constant for the cis form decreased from 0.58 ±
0.02 to 0.34 ± 0.02 ms and that for the trans form reduced
from 12.30 ± 0.41 to 2.01 ± 0.23 ms exponentially with the
voltage increasing from +70 to +110 mV (Fig. 3c and Table S4,
ESI†). The unzipping time constant of the native duplex always
lay between the two isomers of azo-dsDNA, exponentially
dropping from 7.59 ± 0.35 to 0.78 ± 0.08 ms with the rising
potential from +70 to +110 mV. These results not only further
confirmed the unzipping translocation of the duplexes through
the nanopore20 but also showed that the duplex stability of the
trans form and the instability of the cis form relative to the
native one were independent of the potentials. Noted that the
subtle difference of the structural stability was enlarged at low
potential. As shown in Fig. 3c, the τ gap (Δτ) be-tween the cis
form of azo-dsDNA and native duplex decreased from 7.01 to
0.44 ms and that between the trans form and the native one
reduced from 4.72 to 1.23 ms as the potential grew from +70
to +110 mV. This might originate from that the activation
energy of high potential exceeds the unzipping energy barrier
and the unzipping behaviour is not limited by the high
potential. Although low potential allowed the high sensitivity
for detecting the duplex stability, it limited the capture rate of
DNA by the nanopore.24 Thus, +80 mV was chosen for main
analysis in our system.
In summary, we utilized an α-HL nanopore biosensor to
characterize the duplex stability induced by single azobenzene.
The kinetic results of duplex dissociation were acquired by the
nanopore at single molecule level, which have been
interpreted in terms of the structural stability of the duplexes.
Our results provide incontrovertible evidence for that single
trans-azobenzene stabilizes the duplex conformation
dramatically, whereas single cis-azobenzene facilitates the
strand dissociation compared with the native duplex. Further,
the isomerization efficiency between the two isomers of the
azo-dsDNA was calculated at single molecule level. Besides,
the α-HL nanopore biosensor provides a standard for designing
an efficient photo-responsive DNA, which is the narrow width
of the two unzipping time peaks for the isomers and the large
gap between them.
Furthermore, the isomerization efficiency between the trans
and cis form was calculated at single molecule level. Under our
experimental conditions, the isomerization efficiency was (46 ±
3) % for trans-to-cis and (71 ± 7) % for cis-to-trans (Fig. 3d).
These values are comparable to them obtained via the
This research was supported by the National Natural Science
Foundation of China (21505043, 21421004, 21327807) and the
Fundamental Research Funds for the Central Universities
This journal is © The Royal Society of Chemistry 20xx
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(222201718001, 222201717003, 222201714012). Y. L. Ying is
supported by the Chinese Post-doctoral Fund (2016T90340). J.
J. Zhang is supported by the National Natural Science
Foundation of China (21420102004).
Conflicts of interest
There are no conflicts to declare.
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