A Time-Resolved Single-Molecular Train Based on Aerolysin Nanopore

Article abstract of DOI:10.1016/j.chempr.2018.05.004

An aerolysin nanopore interface was introduced as a molecular machine to electrically read out the real-time photo-controlled motion of an azobenzene-geared DNA train with high spatial and temporal resolution. Under alternating UV and visible irradiation, each DNA train performed two regulated speeds of 1.9 and 6.3 bases/s corresponding to trans and cis states, respectively, with readily identified current signals. Each train type fell into the ultra-narrow current population with a full-width half maximum of 0.2–0.4 pA. The combination of a model molecular machine system and powerful aerolysin interface enabled the motions of every artificial molecular machine to be followed in real time. The molecular motion study of a single-molecule machine is essential for excluding the average effect and investigating the mechanistic features of an individual machine at its precise state. To achieve this goal, we developed a small molecular “train” on a nanopore single-molecule interface for the real-time study of the light-regulated motion of a single train. Under alternate UV-visible irradiation, different motions were successfully discriminated at the single-molecule level with ultra-high resolution. Moreover, direct correlation of the motion of molecular machines to time-series readouts and real-time tracing was achieved. A light-switchable single-molecule train was developed on a nanopore single-molecule interface. Upon light regulation, a real-time motion study of a single-molecular machine was achieved. The translocation behavior of trans- and cis-Azo DNA trains through the nanopore platform showed distinctive differences both in speed and in current signal, which could be well discriminated at ultra-high resolution.

Full text of DOI:10.1016/j.chempr.2018.05.004

Article
A Time-Resolved Single Molecular Train Based
on Aerolysin Nanopore
Yi-Lun Ying, Zi-Yuan Li, Zheng-Li
Hu, ..., Chan Cao, Yi-Tao Long,
He Tian
zhangjunji@ecust.edu.cn (J.Z.)
ytlong@ecust.edu.cn (Y.-T.L.)
HIGHLIGHTS
An aerolysin nanopore-based,
photo-controllable single-
molecule machine
Real-time following of two
motions of trans- and cis-Azo-
DNA trains regulated by light
Ultra-high resolution between
trans- and cis-trains could be
discriminated
A light-switchable single-molecule train was developed on a nanopore single-
molecule interface. Upon light regulation, a real-time motion study of a single-
molecular machine was achieved. The translocation behavior of trans- and cis-Azo
DNA trains through the nanopore platform showed distinctive differences both in
speed and in current signal, which could be well discriminated at ultra-high
resolution.
Ying et al., Chem 4, 1–9
August 9, 2018 ª 2018 Elsevier Inc.
https://doi.org/10.1016/j.chempr.2018.05.004

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Article
A Time-Resolved Single Molecular Train
Based on Aerolysin Nanopore
1
1
1,2,
Yi-Lun Ying,1 Zi-Yuan Li,1 Zheng-Li Hu,1 Junji Zhang,1, Fu-Na Meng, Chan Cao, Yi-Tao Long,
*
*
and He Tian1
The Bigger Picture
SUMMARY
The molecular motion study of a
single-molecule machine is
essential for excluding the
An aerolysin nanopore interface was introduced as a molecular machine
to electrically read out the real-time photo-controlled motion of an azoben-
zene-geared DNA train with high spatial and temporal resolution. Under alter-
nating UV and visible irradiation, each DNA train performed two regulated
speeds of 1.9 and 6.3 bases/s corresponding to trans and cis states, respec-
tively, with readily identified current signals. Each train type fell into the ul-
tra-narrow current population with a full-width half maximum of 0.2–0.4 pA.
The combination of a model molecular machine system and powerful aerolysin
interface enabled the motions of every artificial molecular machine to be fol-
lowed in real time.
average effect and investigating
the mechanistic features of an
individual machine at its precise
state. To achieve this goal, we
developed a small molecular
‘‘train’’ on a nanopore single-
molecule interface for the real-
time study of the light-regulated
motion of a single train. Under
alternate UV-visible irradiation,
different motions were
INTRODUCTION
An artificial molecular machine—a self-assembly of several functional molecular
components—has been constructed and devised through inspiration from ma-
chine-like biological molecules working in living cells.1–13 Although many molecular
machines have been presented initially in the solution phase and then on the func-
tionalized surface, investigation of single-molecule motion becomes essential
because each molecule should have specific functions corresponding to its precise
state. The average effect of the random distribution of molecular machines
obscures crucial mechanistic features of the individual machines.14–17 To overcome
this challenge, researchers have recently grafted molecular machines on surfaces
and nano-interfaces where molecules are assigned with biased directions and
arrangements.14–16 More importantly, the direct correlation of the motion of molec-
ular machines to time-series readouts, much less real-time tracing, is still hard to
achieve. Therefore, the study of the single-molecule performance of molecular
machines is still under investigation, probably because of the lack of a proper
nano-interface. The biological nanopore, a general tool for single-molecule analysis,
possesses a well-confined space for accommoding individual molecules, which
offers the possibility to develop studies of single-molecule machine motion with
continuous electric readouts. Previous studies demonstrated that nanopores could
act as a powerful new frontier in the study of single-molecule dynamic behaviors
with high spatial and temporal resolution.18–20 The translocation of single molecules
through nanopores has been regarded as a man-made transmembrane molecular
machine.21 For example, an a-hemolysin nanopore has been introduced for studying
host-guest self-assembly and photo-responsive stereo-interconversion in manufac-
tured supramolecular systems or even monitoring weak molecular interactions.22–25
Compared with conventional spectroscopy and microscopy, the nanopore interface
offers the possibility of real-time following of single-molecule motion rather than
focusing on the initial or ending states.26,27
successfully discriminated at the
single-molecule level with ultra-
high resolution. Moreover, direct
correlation of the motion of
molecular machines to time-series
readouts and real-time tracing
was achieved.
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However, the overlap of signals between different motion states still exists in
nanopore studies,24,25 which severely hampers the discrimination of different work-
ing states as well as the precise control of single-molecule machine systems. In our
previous research, we used an aerolysin nanopore to resolve individual short
polydeoxyadenines (dAn).28,29 Aerolysin shows high capacity for size discrimination
of polyethylene glycols,30 the detection of peptides,31 and the identification of
single-amino-acid differences in peptides32 and proteins33 and protein-DNA
chimera transport.34 Recently, an aerolysin nanopore was used for identifying the
configuration of photo-responsive oligonucleotides.35 Moreover, biological nano-
pores have a promising application in the characterization of molecular motors
and revealing the dynamic interactions between biomolecules.36,37 Because the
ꢀ1 nm constriction of aerolysin38–40 matches the size of most artificial molecular ma-
chines, this aerolysin nanopore provides us with a stereo-confined nano-interface to
significantly reduce the randomness of single-molecule motions and promote a
high-resolution analysis platform for the study of individual machines. Here, we pre-
sented a ‘‘photo-geared’’ oligonucleotide ‘‘molecular train,’’ 50-AA-azobenzene-
AA-30 (Azo) single-stranded DNA (ssDNA), on an aerolysin nano-interface.
The photo-triggered individual train motions of the two isomeric states were
well discriminated and studied in real time via time-resolved current signal readouts
(Figure 1 and Figure S1).
RESULTS
The azobenzene-modified single-strand oligonucleotide (Azo-ssDNA) was synthe-
sized according to a previously reported protocol (see Supplemental Experimental
Procedures). The synthesized azobenzene moiety was introduced into poly(dA)4
through a D-threoninol linker. Photo-isomerization performance was first investi-
gated in buffer solution (see Figure S2), which revealed good cis-to-trans intercon-
version of the designed Azo-ssDNA train. Aerolysin, produced by Aeromonas sp.,
could self-assemble from a monomer into a heptameric pore-forming protein.38
A near-atomistic structure implied that the rivet-like aerolysin may process the nar-
row bottlenecks of approximate 1 nm at its extracellular mouth or transmembrane b
barrel.39,40 In our study, an aerolysin nanopore was embedded into a lipid bilayer
across which +100 mV was applied via a pair of Ag or AgCl electrodes (Figure 1).
The two chambers beside the bilayer contained 1 M KCl buffered with 10 mM Tris
and 1 mM EDTA (pH 8.0). In the first step, the 2.0 mM Azo-ssDNA (mainly in its trans
form) irradiated with visible light (l > 450 nm) was added to the cap side of the pore
under a dark environment (see Figure S3). Visible-light irradiation before the addi-
tion of the sample was to ensure that most of the Azo-ssDNA was in its trans form.
Note that the constant I–V curves indicated that aerolysin maintained an intact
conformation under ultraviolet-visible (UV-vis) irradiation (see Figure S4). As an
Azo-ssDNA train enters the aerolysin, it interrupts the ionic current through the chan-
nel at an applied potential of +100 mV, resulting in a blockage signal (Figure 1, left).
According to a previous study,28 an oligonucleotide traversing the aerolysin induces
a duration longer than 0.2 ms, and it collides with the cap opening of the pore
and yields short-lived blockages with duration shorter than 0.2 ms. For example,
1Key Laboratory for Advanced Materials & School
a dA4 traversing an aerolysin produces a duration of 9.2 ms and I/I0 = 0.52 at
+100 mV (see Figure S5). Here, I0 refers to the open pore current, and I represents
the residual current caused by the DNA. In order to focus on the translocation
behavior of each individual molecular train, we excluded all collision blockages for
further analysis. The two-dimensional (2D) scatter plots of the visible-light-irradiated
Azo-ssDNA train yielded two characteristic populations with Gaussian-fitted peak
values of I/I0 = 0.25 and 0.31 (Figure 2A). Compared with the previously reported
of Chemistry and Molecular Engineering, East
China University of Science and Technology,
Shanghai 200237, P.R. China
2Lead Contact
*Correspondence:
zhangjunji@ecust.edu.cn (J.Z.),
ytlong@ecust.edu.cn (Y.-T.L.)
https://doi.org/10.1016/j.chempr.2018.05.004
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Figure 1. Schematic Representation of Time-Resolved Electric Current Readouts of Individual Photo-Reversible 50-AA-azobenzene-AA-30 Based on an
Aerolysin Nano-interface
(Top) The synthesized azobenzene moiety is introduced into poly(dA)4 through the D-threoninol linker. The azobenzene group in the oligonucleotide is
highlighted in dark blue, and it isomerizes between trans and cis states under UV-vis light.
(Middle) The speed-regulated molecule train of 50-AA-azobenzene-AA-30 traversing an aerolysin nanopore from its cap side. The UV light induces the
high speed of designed cis-Azo-ssDNA, whereas the visible irradiation prolongs the translocation of trans-Azo-ssDNA. The illustration is not to scale.
(Bottom) Electric recording of 50-AA-azobenzene-AA-30 with an aerolysin nanopore. The current blockage of trans-Azo-ssDNA has a longer duration and
deeper blockage amplitude than that of cis-Azo-ssDNA.
translocation of dA5 through aerolysin (I/I0 = 0.40), a deeper current blockage of
Azo-ssDNA might be due to increased bulkiness of the oligonucleotide side chain
after azobenzene modification.
Interestingly, the number of blockages in the population of I/I0 = 0.31 is around
six times smaller than that in the population of I/I0 = 0.25 (Figure 2A). Because the
Azo-ssDNA isomers present mainly in trans form under visible light, we suggest
that trans-Azo-ssDNA induced the larger population of I/I0 = 0.25 (trans-train); small
amounts of cis-Azo-ssDNA might contribute to this smaller population of I/I0 = 0.31
(cis-train) in view of the dynamic feature of photo-switchable azobenzene. To prove
our speculation, we then imposed UV light (l = 365 nm) on the chamber containing
trans-Azo-ssDNA. As expected, the increased numbers of cis-trains were obtained
identically at I/I0 = 0.31 without the generation of other obvious translocation pop-
ulations. These results further support the assignment of two characteristic current
populations, which could be used for determining the reversible interconversion
between trans- and cis-Azo-ssDNA.
To our surprise, a current separation was obtained between the trans- and cis-Azo-
ssDNA trains. Under alternating UV and visible irradiation, the full widths at half
maximum (FWHMs) of the Gaussian peaks for the trans-train and cis-train displayed
I/I0 values of 0.004 and 0.008, which correspond to I values of 0.2 and 0.4 pA, respec-
tively. Therefore, the blockages from both trans- and cis-trains fell into sharp
Gaussian distributions with FWHMs as narrow as 0.2 and 0.4 pA, respectively. The
Gaussian peak separation is the difference between two adjacent Gaussian peak
values, which is ꢀ6% between two peaks for trans- and cis-trains. Therefore, almost
no overlap was observed between the Gaussian peaks (Figure 2). In our previous
work, good Gaussian peak separation of 10%–15% was able to discriminate the
length dependence of dAn (n = 2, 3, 4, and 5). The current separation of dAn varies
depending on the mass differences of single deoxyadenosine monophosphate (mo-
lecular weight = 331.22). However, in our molecular train system, the difference be-
tween the two photo-isomer states was merely from the isomerization of one
‘‘pseudo-nucleic base’’ (azobenzene) with identical molecular mass. Therefore, our
study suggests that the aerolysin nanopore not only is capable of mass discrimina-
tion but also shows a superior ability for steric identification of oligonucleotides. Pre-
˚
vious studies have suggested that the trans-azobenzene (9.0 A in length) is favorable
for protruding from the backbone of ssDNA.41,42 On the other hand, the cis-azoben-
˚
zene (5.5 A in length) is prone to forcing itself to fit in the gap between neighboring
bases.41,42 Therefore, the trans-Azo-ssDNA train exhibits greater steric hindrance of
ionic flow through the aerolysin nanopore than the cis-Azo-ssDNA train, resulting in
a deeper blockage. With these encouraging results, we conclude that blockages of a
single Azo-ssDNA molecular train in both trans- and cis-isomer states could be well
discriminated with high current resolution with aerolysin nanopores.
As shown in Figure 2A, an ultra-long duration time (tD = 2,630.3 ms) was observed for
the trans-train. The translocation speed was calculated as 1.9 bases/s, which is over
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Figure 2. Statistical Analysis of the Translocation Events of Azo-ssDNA
Translocation events were analyzed under visible (A) and UV (B) irradiation. The 2D contour plots are composed of 2,000 blockage events. The two
populations in the 2D contour plots correspond to two Gaussian peaks in the current histogram (top), and their duration histogram on a log scale is fitted
into Gaussian functions (right). In the current and duration histograms, the population for the trans-train is highlighted in yellow, and that for the cis-train
is shown in purple. The data were acquired in 1 M KCl, 10 mM Tris, and 1 mM EDTA (pH 8.0) at +100 mV in the presence of 2.0 mM Azo-ssDNA.
500 times slower than that of previously reported polydeoxyadenines (dA5).28 The
introduction of azobenzene as a pseudo-nucleic base, on the one hand, successfully
performed as a gear in the DNA train, which decelerated its translocation speed. On
the other hand, the cis-train showed a higher speed of 6.3 bases/s (tD = 794.3 ms,
Figure 2B), almost 3.3 times faster than the trans-train. As mentioned before, cis-
azobenzene presents less steric hindrance than its trans counterpart, which leads
to a faster translocation speed. A previous study highlighted that the sensing capa-
bility of aerolysin is attributed to the electrostatic interaction between the positively
charged residues positioned in the b barrel and the negatively charged oligonucle-
otides.39 Because the protruded long side chain of trans-Azo-ssDNA offers nearly
full occupation (I/I0 = 0.25), the crowded environment in the pore lumen largely in-
creases the possibility for electrostatic interactions between pore and train, leading
to a slow translocation. Moreover, the flipping out of trans-azobenzene ensures the
probability of its interacting with the aromatic residue inside the lumen of aerolysin.
Therefore, the gearing effect and photo-regulated translocation speeds of cis- and
trans-Azo-ssDNA are closely related to its structure and the interactions with the
pore.
The above electric current recording indicates that photo-gearing of single trans-
and cis-Azo-ssDNA trains could be precisely converted into time-resolved
blockages. Therefore, we further investigated the reversible gear shift of a single
Azo-ssDNA train in aerolysin in real time (Figure 3A). After adding Azo-ssDNA to
the cap side of the aerolysin nanopore, we detected dominant long-time trans-train
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signals (I/I0 = 0.25) in the first 5 min under a dark environment, indicating the initial
major trans state of the DNA train (see Figure S7). UV light (l = 365 nm) was then
applied in situ, and short-lived cis-train blockages (I/I0 = 0.31) gradually appeared
with a consequent decrease in the initial trans-train blockages (I/I0 = 0.25). The
trans-/cis-train signal ratio reached saturation (0.4:1) after 15 min of UV irradiation
(Figures 3B and 3C), presenting photo-isomerization or, in other words, a trans-cis
gear shift of the photo-responsive Azo-ssDNA train. Note that the cis-trans gear-shift
process could be read out immediately after the light source was changed from UV
(l = 365 nm) to visible (l > 450 nm) light. After 20 min re-illumination of visible light,
the long-time trans-train signals were regenerated and gradually took the lead with
the saturation ratio (Ntrans-train/Ncis-train) of 5.8:1 (Figure 3C).
To clearly demonstrate the real-time photo-switching processes as well as the
single-molecule analysis capability, we presented the I/I0 of each trans- and cis-train
blockage with the function of continuous irradiation time (tR). Each dot in Figure 3B
represents the individual translocation motion of one specific Azo-ssDNA train. We
recorded every irradiation cycle after 17 min under UV and 20 min under visible light
to ensure saturated photo-isomerization. The I/I0 value for the center of the upper
population was 0.31, and that of the lower population was 0.25, which represent
cis-Azo-ssDNA and trans-Azo-ssDNA, respectively. With the ongoing UV irradiation
time, the event density of the cis train increased continuously as that of trans-train
became sparse; the inverse process occurred when visible light was subsequently
imposed. In contrast to free azobenzene molecules in solution, azobenzene nucleic
bases tethered in the DNA strand should overcome the steric hindrance from
adjacent adenosine bases to perform photo-induced conformation changes. This
would lead to lower isomerization efficiency and incomplete conversion during
UV-vis irradiation.43 Because aerolysin nanopores provide high resolution for
discriminating each train’s blockage, Figure 3B reveals that not all the translocation
events could be accurately assigned to trans- or cis-trains. Thus, the sporadic block-
ages distributed around the two Gaussian populations may originate from those
sub-states during isomerization, such as incomplete conversion or photo-intermedi-
ates, photo-conversion-induced misfolding of DNA strands, or even unexpected
photo-byproducts under light excitation. The number of blockages for the trans-
train and cis-train are represented as Ntrans-train and Ncis-train, respectively. The value
of Ntrans-train/Ncis-train was evaluated after the photo-isomerization reached
saturation, which demonstrates the reversible conversion of the trans- and cis-
train blockages (Figure 3C). Moreover, several cycles of real-time photo-induced
interconversion between trans- and cis-train blockages were conducted with con-
stant values of I/I0 and duration times for both trans-Azo-ssDNA and cis-Azo-ssDNA
(Figures 3D and 3E), demonstrating the high reproducibility of our design.
DISCUSSION
In summary, the photo-controlled motion of an Azo-ssDNA molecular train at the
single-molecule level was achieved on the basis of an aerolysin nano-interface.
The time-resolved electric readouts revealed that the trans-train (slow) and cis-train
(fast) exhibited distinct current blockages with 3.3 times the difference for photo-
geared speeds. More importantly, both the translocation motions of a single-
molecule train were able to be monitored in real time, demonstrating the high
spatial and temporal resolution of our nanopore interface for precisely conducting
single-molecule machine behavior. Moreover, sub-states events could also be de-
tected during the molecular train motion, providing guidance for the improvement
of accurate molecular machine operation. Compared with previous molecular
6
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Figure 3. Real-Time Discrimination between the Motion of Single trans- and cis-Azo-ssDNA Trains through an Aerolysin Nano-interface
(A) The real-time electric current readouts of Azo-ssDNA under UV-vis irradiation. The purple represents cis-train blockages, and the yellow indicates
trans-train blockages. The initial part of the UV irradiation current trace was acquired after continuous irradiation with UV light (l = 365 nm) for 15 min.
Then, the irradiated wavelength changed to l > 450 nm at irradiation time for visible light (tR-vis) of 0 min. The following representative current traces
were recorded at UV irradiation times of 5 and 10 min.
(B) Reversible effect of UV-vis light on the regulation of cis- and trans-trains. The corresponding contour plots are shown in Figure S6. The widths of the
two bands were calculated according to the peak width of the individual Gaussian current fits. The number of trans- and cis-train blockages varied from
the initial state to the final saturation under visible and UV illumination, respectively.
(C) Reversible conversion between the number of trans-trains and cis-trains induced by UV-vis light. The number of blockages for the trans-train and
cis-train are represented as Ntrans-train and Ncis-train, respectively. The value of Ntrans-train/Ncis-train was evaluated after the photo-isomerization reached
saturation. Therefore, the nanopore data were collected in the dark after UV or visible illumination for 17 or 20 min, respectively.
(D) The high reproducibility of the durations for the trans-train and cis-train under a continuous irradiation cycle.
(E) The high repeatability of I/I0 for the trans-train and cis-train under a continuous irradiation cycle.
machines on gold surfaces and silica nanoparticles,9 the nanopore interface offers a
platform for device application of a single-molecule machine. In future, more elab-
orately designed molecular machine systems could be fabricated on the basis of the
nanopore interface to perform multiple tasks. For example, with the development of
nanopore arrays, single-molecule computing would be possible because each
nanopore molecular machine performs a logic operation corresponding to different
external stimuli. These molecular machines might never replace conventional
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silica-circuitry-based computers; however, they have great potential in biological
and physiological computing systems as well as in vivo applications.
EXPERIMENTAL PROCEDURES
Detailed experimental procedures, including the preparation of aerolysin nano-
pores, electrochemical measurements, data analysis, and compound synthesis,
are provided in the Supplemental Information.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures and
eight figures and can be found with this article online at https://doi.org/10.1016/j.
chempr.2018.05.004.
ACKNOWLEDGMENTS
This research was supported by the National Natural Science Foundation of China
(21421004, 21327807, and 21505043) and the Fundamental Research Funds for
the Central Universities (222201718001, 222201717003, and 222201714012).
AUTHOR CONTRIBUTIONS
Y.-L.Y., J.Z., H.T., and Y.-T.L. designed the research; Z.-L.H., Z.-Y.L., and F.-N.M.
performed the research; Z.-Y.L. and J.Z. synthesized the compounds; Y.-L.Y.,
Z.-L.H., and Z.-Y.L. analyzed the data; and Y.-L.Y., J.Z., C.C., Y.-T.L., and H.T. wrote
the paper.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: February 11, 2018
Revised: April 16, 2018
Accepted: May 7, 2018
Published: June 14, 2018
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