DOI: 10.1002/cphc.201700982

Minireviews

Synthetic Ion Channels and DNA Logic Gates as
Components of Molecular Robots
Ryuji Kawano*[a]A molecular robot is a next-generation biochemical machinethat imitates the actions of microorganisms. It is made of biomaterials such as DNA, proteins, and lipids. Three prerequisites
have been proposed for the construction of such a robot: sensors, intelligence, and actuators. This Minireview focuses on
recent research on synthetic ion channels and DNA computing
technologies, which are viewed as potential candidate components of molecular robots. Synthetic ion channels, which areembedded in artificial cell membranes (lipid bilayers), sense
ambient ions or chemicals and import them. These artificial

sensors are useful components for molecular robots withbodies consisting of a lipid bilayer because they enable the interface between the inside and outside of the molecular robot
to function as gates. After the signal molecules arrive inside
the molecular robot, they can operate DNA logic gates, which
perform computations. These functions will be integrated into
the intelligence and sensor sections of molecular robots. Soon,these molecular machines will be able to be assembled to operate as a mass microrobot and play an active role in environmental monitoring and in vivo diagnosis or therapy.

1. Molecular Robots and the Lipid Bilayer Platform
Molecular robots have recently emerged based on biomolecules and biochemical processes. Approximately 30 years ago,
a self-constructing machine, a so-called “assembler,” was originally proposed by Drexler.[1] Based on the idea of an assembler,
molecular machines that operate autonomously have been developed using DNA or RNA. For example, DNA walkers moveautonomously, on the basis of energy supplied from the hybridization of fuel oligonucleotides, from one binding site toanother on a DNA-modified surface.[2, 3] Rothemund has also
proposed a method by which DNA molecules can be folded
into any desired two-dimensional shape to make “DNA origami.”[4] In the field of synthetic chemistry, nanosized machines
such as motors and ratchets have been developed based onorganic or supramolecular chemistry.[5–7] This was viewed as
ground-breaking technology and the pioneers have since beenhonored with the Nobel Prize in Chemistry 2016.
Inspired by the idea of a molecular assembler, molecular robotics, which involves construction with a much higher dimension of assembly, was proposed in 2014.[8] Molecular robots arecomposed of sensors, calculators, and actuators that are all implemented in liposomes or hydrogels. White blood cells, themost imageable example, senses the chemical signals secreted
from a target bacterium, calculates the direction and length
between the target and itself, and moves toward the signal

source by chemotaxis. These accomplished functions are integrated in a micron-sized body surrounded with a bilayer lipid
membrane (BLM). Sato et al. reported the development of a
sophisticated molecular robot prototype in 2017.[9] Their developed amoeba-type robot has light-induced DNA clutches forsensors and kinesin-microtubule proteins as actuators, all integrated in a cell-sized liposome. Light irradiation acts as a trigger for the release of the signal molecules and disengagementof the DNA clutches to change the shape of the liposome.
The fabrication process used for existing mechanical robots
is viewed as a blueprint for the manufacture of these molecular robots. In the case of humanoid robots, the arms and legs
are manufactured individually and then assembled. Similarly,
individual fabrication would be a straightforward process inthe manufacturing of molecular robots. Hence, a prototyping
factory is required, as well as an industrial manufacturing process.
In this Minireview, I focus on manufacturing the body of themolecular robot using BLM with membrane receptors(Figure 1). BLM is the ideal material for the body of molecularrobots because it is naturally biocompatible and can host receptor proteins. In addition, physical dynamics such as membrane fusion and endo- or exocytosis, such as molecular
uptake processes, are useful for the interface of the robots. We
previously developed a high-throughput planar BLM (pBLM)
system that will be a powerful tool for the manufacturing of
the lipid body of molecular robots.[10–12] A pBLM is generally
used as the ion current measurement of an ion channel orpore-forming proteins. Because the reproducibility and stabilityof pBLMs are conventionally insufficient, various methods havebeen proposed to overcome this, primarily using microfluidic
technology.[13] The most promising method is the droplet contact method,[14, 15] in which two microdroplets surrounding the[a] Dr. R. Kawano
Department of Biotechnology and Life Science
Tokyo University of Agriculture and Technology (TUAT)
2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588 (Japan)
E-mail: rjkawano@cc.tuat.ac.jp
The ORCID identification number for the author of this article can befound under: https://doi.org/10.1002/cphc.201700982.
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DNA computing technology with nanopore proteins will beable to connect the sensor and intelligence functions.

2. Synthetic Ion Channels and Transport
Control at the Lipid Bilayer Membrane
Receptor proteins sense specific molecules in the cell membrane. When the ligand molecule binds to a receptor, the receptor senses the binding and produces a signal that is transmitted downstream to control ion transport through ion channels, as it cascades. Furthermore, pore-forming proteins play a
key role in the transportation of molecules with the gradientof membrane potential or the substrate concentration. Artificial
ion channels or pores have been created using synthetic
chemistry to mimic the structure or function of natural proteins.[19, 20] In the last three decades, synthetic chemists haveproposed various compounds that actively exhibit ion channel
structures and functions.[21] As part of the design, chemists try
to add functions such as pore size, ion or substitute selectivity,and voltage or ligand gating. In the early studies, researchers
imitated natural compounds, such as valinomycin or gramicidin, for the structural framework of synthetic channels (Figure 2 a). Valinomycin is a macrocyclic polypeptide used in thetransport of potassium, as an antimicrobial peptide. Gramicidin
is also a polypeptide and forms a b-helix structure in the lipid
monolayer;[22] within the bilayer, two gramicidin molecules
form an end-to-end dimer, which in turn forms a transmembrane structure in the bilayer. These macrocyclic or dimer
structures have been modeled for the design of synthetic
channels, and chemists synthesized the mimicking structureusing synthetic peptides or macromolecules. More recently,
other functional materials such as DNA origami and carbon
nanotubes have been proposed as potential candidates for artificial ion channels.[19, 23, 24]
One of the most sought after properties is ion selectivity for
Na + , K + , Ca2 + , Cl@ , and other ions. Most studies have focusedon synthesis of the channel structure in the lipid membrane,
and assessed the ion selectivity. Among them, the first reportof a selective channel was for K + ions.[25] In nature, potassiumchannel proteins use a selective filter that has five amino acids,
TVGYG, within each of the four subunits. The hydrated K + ion
is dehydrated by interacting with these motifs, and thenpasses through the filter pore with high ion selectivity(Figure 2 b).[26] Conversely, in the synthetic channels, theK + -selective filter is composed of an aromatic ring (primarily
formed by four tyrosine residues) that provides a weak electricfield, allowing complete dehydration of K + ions, but not of
Na + ions.
In this system, the p electrons of the aromatic rings contribute to the lowering of the potential barrier for passage of K +ions through attractive p–cation interactions (Figure 2 c).[20] Following examination of K + selectivity, the selection trend, forexample, Li + > Na + > K + , is estimated in terms of alkali metal
ions, as the Eisenman sequence.[27] The selectivity for otherions or molecules, such as halide ions, divalent ions, and ammonium ions has also been studied extensively by researchers.
More recently, a de novo strategy from protein engineering

Figure 1. Conceptual illustration of a molecular robot. The body consists ofa lipid bilayer with synthetic ion channels as the gate. DNA computing architecture is integrated inside the robot as the intelligence. (This illustration isused courtesy of Professor S. Murata of Tohoku University).

lipid monolayer are brought into contact and a stable and reproducible pBLM is formed at the droplet interface, as thedroplet–interface bilayer. This method is simple and rapid, andthe formed pBLM is extremely stable. Based on this method,
several high-throughput pBLM formation methods have been
proposed and applied to large-scale measurement of ion channels or nanopore measurements.[16–18]
In the following section, two recent efforts are introduced
that attempted 1) to use a synthetic ion channel as the artificial receptor protein, and 2) autonomous sensing and calculation using DNA computing with nanopore technology using ahigh-throughput pBLM system. Synthetic channels are potential candidates for the sensor sections of molecular robots.

Ryuji Kawano was born in Oita, Japan
in 1976. He received his Ph.D. in 2005
from Yokohama National University
under the supervision of Prof. Masayoshi Watanabe, working on dyesensitized solar cells using ionic liquid
electrolytes and analyzing the chargetransport mechanism using electrochemistry. Subsequently, he spent approximately three years in Prof.
Henry S. White’s laboratory at the University of Utah as a postdoctoral researcher, where he conducted research on nanopore measurements with glass materials. He then joined Prof. Shoji Takeuchi’sgroup at the Kanagawa Academy of Science and Technology(KAST) and University of Tokyo, where he carried out research on
the construction of durable lipid bilayer systems using microfabricated devices. Since 2014, he has been working as an Associate
Professor (tenure-track) at Tokyo University of Agriculture and Technology. His current research interests include molecular robotics
based on synthetic membrane proteins, DNA computing, andnanopore technology based on microfabrication.

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Figure 2. Natural and synthetic ion channels. a) Structures of valinomycin and gramicidin. Gramicidin is a toxin channel and forms a transmembrane structure
by dimerization.[22] b) Structure of the K + -selective filter of the KcsA channel (deposited in the Protein Data Bank under accession no. 1K4C). The side chain of
the amino acid residue threonine of the signature sequence is shown because it participates in coordinating a K + ion at site S4.[26] c) Schematic representationof the selective filter of a general synthetic channel. The filter consists of aromatic rings and cation–p interactions are important for selectivity.[20] Reproducedwith permission from Ref. [20]. Copyright (2006) Royal Society of Chemistry. d) Structure of a transmembrane Zn2 + -selective channel, which consists of fourhelix bundles of de novo designed short polypeptides.[28] Reproduced with permission from Ref. [28]. Copyright (2014) The American Association for the
Advancement of Science.

has become a powerful tool for designing ion-selective peptide channels. DeGrado and co-workers reported on the designof a membrane-spanning, four-helical bundle peptide thattransports Zn2 + and Co2 + ions, but not Ca2 + , across membranes (Figure 2 d).[28]Other challenging directions of synthetic channel studies areactive transportation (pumping) and control of open–close
states (gating). Although these two properties (i.e., pumping
and gating) are suitable for imitating natural ion channels, thestrategies of the imitation are still being explored. In the context of pumping, the transport of ions across membranes andagainst a thermodynamic gradient is essential to many biological processes. Gust et al. proposed a strategy for photo-induced Ca2 + ion transport via lipid membranes.[29] The proposed strategy uses a synthetic, light-driven transmembrane
Ca2 + pump based on a redox-sensitive lipophilic Ca2 + -binding
shuttle molecule powered by an intramembrane artificial photosynthetic reaction center (Figure 3 a). The active transport isdriven not by concentration gradients, but by light-induced
electron transfer in a photoactive center that is asymmetricallydisposed across a lipid bilayer. Photoswitchable molecules for
manipulating ion channels have also been proposed by Trauner and Feringa.[30, 31] Other interesting strategies based onelectron transfer with a redox reaction have also been proposed. Stoddart and co-workers synthesized a wholly artificial
supermolecule that acts on small molecules to create a gradient in their local concentration (Figure 3 b).[32] This enables aredox-active ring viologen to move along an oligomethylene
axis as the redox reactions proceed. In experiments conducted,
the artificial pump operated reversibly for two cycles of operaChemPhysChem 2018, 19, 359 – 366

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tion and drove rings against the concentration gradient, but
the system did not conduct movement in the lipid membrane.
Controlling the open-close state (gating) of synthetic channels, by contrast, is very challenging. Most approaches utilize
voltage gating: an early approach used by Fyles and co-workers[33] and Kobuke and co-workers[34] involved an asymmetricalstructure with charged parts at one of the termini in the transmembrane molecules. Kawano, Furukawa and co-workers havealso attempted to create gating-controllable synthetic channels.[35] They focused on metal–organic scaffolds because of
the tunability of the structure.[36] Metal–organic frameworks
have attracted substantial attention as porous materials, especially for adsorption or storage of gas molecules. Recently, Kim
and co-workers[37] investigated the capability of metal–organic
molecules as synthetic ion channels. Following their study, we
synthesized rhodium metal–organic polyhedrals (RhMOPs) thathave two different lengths of alkoxy chains (C12 and C14) at the
periphery for controlling the open-close state, called
C12RhMOP and C14RhMOP.[35] In addition, these RhMOP molecules showed two distinct channel conductance states because
the Archimedean geometry of the MOP structure allows
porous molecules to possess more than two different polygonal apertures and one internal cavity (Figure 3 c). Therefore,
ions pass through the internal cavity of the RhMOP via either
the square or the triangular aperture exposed to the aqueous
phase in the lipid bilayer. The long alkoxy chains at the periphery can facilitate interaction with lipid molecules, and the difference in the length enables modulation of its interaction
such that it can alter the molecular dynamics, resulting inswitching between the open–close states.
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Figure 3. Synthetic ion channels with particular functions. a) Schematic illustration of a light-powered transmembrane Ca2 + pump system.[29] Reproduced withpermission from Ref. [29]. Copyright (2002) Nature Publishing Group. b) Structure and schematic illustration of the molecular pump that operates the redox
reaction.[32] Reproduced with permission from Ref. [32]. Copyright (2015) Nature Publishing Group. c1) Schematic illustration and channel signals of the open–
close state of the MOP channel. c2) The multiple conductance states observed from a single molecule. c3) The open-channel state (duration) can becontrolled by changing the outer structure.[35] Reproduced with permission from Ref. [35]. Copyright (2017) Elsevier.

As described above, synthetic channels will be useful tools
at the interface of the molecular robots and their external environment. This is because the selectivity of information, as ionsor molecules, and controllability of the gating are absolutely
imperative. The next step is regulation of these functions fromexternal stimulation using light irradiation, magnetic fields, orelectrical stimulation.

information about individual molecules in terms of size andmobility (Figure 4 a). Although this method can detect single
molecules, the selectivity relies on the size compatibility between the nanopore and target molecules. An a-hemolysin(aHL), pore-forming toxin from Staphylococcus aureus, is commonly used as the biological nanopore. This protein has a
1.4 nm diameter pore that allows single-stranded DNA (ssDNA)
to pass but blocks double-strand DNA (dsDNA), suggestingthat the nanopore has precise size selectivity for DNA/RNA detection.[40, 41] For the detection of smaller molecules, a molecular adapter such as cyclodextrin, which reduces the pore size,has been used (Figure 4 b).[42, 43] DNA aptamers have also been
applied as the molecular tag for selective detection because
aHL cannot detect larger sized molecules (Figure 4 c).[18] Conversely, we have studied much larger nanopores from five different protein families for precise nanopore detection.[44] Recently, a promising application of nanopore measurements for
DNA sequencing has also resulted from ardent efforts, with a

3. Nanopore Technology Meets DNA Computing and is Applied to Practical Sensing
Nanopores—pore-forming transmembrane proteins—are another strong candidate for the sensors of molecular robots.
Sensing with nanopores has emerged as a method for singlemolecule detection.[38, 39] This method works by applying anelectric potential that causes single molecules to be passed
through the nanopore. The change in the ionic current overtime is recorded, potentially allowing the direct collection of
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Figure 4. Nanopore measurements. a) Conceptual illustration of nanopore sensing.[39] Reproduced with permission from Ref. [35]. Copyright (2017) Elsevier.
b) A cyclodextrin embedded in an aHL pore operates as the adapter for detecting small molecules.[42, 43] Reproduced with permission from Ref. [43]. Copyright(2000) Elsevier. c) DNA aptamers are also used for selective detection of small molecules.[18] In the absence of target molecules, the aptamer is a ssDNA andpasses through the pore. In the presence of the target, the DNA aptamer binds it and forms a complex, which cannot pass through the pore, generating
characteristic current signals. Reproduced with permission from Ref. [18]. Copyright (2011) The American Chemical Society.

nanopore sequencer being commercialized in 2015. To thisend, because conventional nanopores are highly compatiblefor detecting oligonucleotides, we have attempted to integrate
the nanopore method into DNA computing in order to connect the sensoria and the intelligence of the molecular robots.
We have attempted to perform NAND logic calculations in afour-droplet system and to detect the output DNA moleculeusing an aHL nanopore reconstituted in the droplet-interface
bilayer (Figure 5 a).[45] This droplet system has two inputs, anoperation droplet for calculations, and an output droplet. Aftercalculating the two input DNAs, they pass through the aHL
nanopore and are transferred from the calculation to the
output droplet. In this operation, ssDNA translocation throughthe aHL nanopore signifies an output of 1, whereas absence of
DNA translocation signifies an output of 0. Output DNA molecules are detected by aHL nanopores with single-molecule
translocation, and the system was label-free. The operation isrelatively faster (approximately 10 min) than the conventional
method. Next, we attempted to integrate more complex operations into this nanopore-droplet system. This system functions
as an AND gate with amplification and transcription from DNA
to RNA, using T7 RNA polymerase (T7RP).[46] To construct thissystem, we used two input DNAs and template DNA containing part of the T7RP promoter region, as shown in Figure 5 b.
For cases in which two input DNAs exist, defined as input (1 1),
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the two DNAs form a duplex that hybridizes with the templateDNA. The T7RP polymerase binds to the promoter region andsynthesizes a large amount of RNA as output 1. In the cases of
inputs (0 0), (0 1), and (1 0), the input DNA cannot hybridize
with the template DNA, resulting in an output of 0. In addition,
this AND gate operation was simultaneously conducted using
a parallel droplet device with a short period (Figure 5 c). Integration of DNA logic gates into electrochemical devices is important to ensure that molecules containing output information, such as diagnostic results, can be processed as humanrecognizable information. In the next step, the programmablesystem is applied to practical application, for example, cancer
diagnosis using microRNA.
MicroRNAs (miRNAs) are noncoding, small, single-stranded
RNAs. They are involved in the regulation of over 60 % ofhuman genes, and they play significant roles in physiologicalprocesses.[47] Accumulated evidence has revealed that aberrant
levels of miRNA expression in tissues or blood is associatedwith various human diseases such as cancers and cardiovascular diseases. Therefore, miRNA is an emerging class of clinicallyimportant biomarkers for early diagnosis. A landmark reporton miRNA detection using aHL nanopore was presented by
Gu and co-workers. They used an oligonucleotide probe thatbinds to the target miRNA and generates programmable current signatures in the nanopore measurements (Figure 6 a).[48]363

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Figure 5. DNA logic gate integrated into the droplet bilayer system. a) NAND operation system with four droplet networks comprising two input, one operation, and one output droplet.[45] b) DNA to RNA transcription AND gate using an enzymatic reaction.[46] Reproduced with permission from Ref. [46]. Copyright(2017) The American Chemical Society. c) Transcription AND operation performed in parallel with the droplet device. c1) Photograph of the device. c2) The operation with the enzyme-catalyzed reaction can be implemented in the droplet. c3) The asymmetric two-solution (buffer-enzyme) condition is key to the enzymatic reaction and nanopore measurement. Reproduced with permission from Ref. [46]. Copyright (2017) The American Chemical Society.

Using this system, they were able to selectively detect miRNAs
at sub-picomolar levels in samples obtained from lung cancer
patients. Subsequently, they applied the method to multiple
miRNA detection by using DNA and polyethylene glycol tags
as the barcode.[49] The system also represented the programmable nanopore currents from four lung-cancer-derived
miRNAs. We also proposed a theranostics system, which involves the combination of diagnosis and therapy at the sametime, using a nanopore-droplet device (Figure 6 b).[50] The proposed system includes autonomous diagnosis of cancers using
miRNA (as the input molecule) and therapy for the tumor cellsby a DNA antisense drug (as the output molecule). In the presence of miR-20a secreted from a small-cell lung cancer (SCLC),
two programmable DNAs functioning as diagnostic molecules
bind to the input miR-20a, and form a three-way junction
structure. At that time, isothermal reactions with enzymes (aKlenow fragment as the polymerase and Nt.AlwI as the nicking
enzyme) repeatedly occur to generate a DNA drug called oblimersen. The generated molecules were quantified by thetranslocation frequency of the nanopore measurement in real
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time (Figure 6 c). The results of nanopore quantification
showed that oblimersen was amplified more than 20-fold fromthe input miR-20a, which meets the dosage requirement for
SCLC therapy. Based on these DNA computing techniques, wehave recently reported an approach for detection of miRNA at
ultra-low concentrations, in which the target miRNA is amplified from 1 fm to pm level and then nanopores with asymmetrical electrolyte condition are detected, which can increase theevent frequency of miRNA translocation.[51]Combining nanopore and DNA computing technologies isuseful for constructing smart and autonomous sensing at theinterface region. The nanopore/DNA computing system willcontribute to the construction of intelligent sensors for molecular robots.

4. Perspectives for Molecular Robots with
Lipid Bilayers
This Minireview focused on functionalization of lipid bilayers
embedded with synthetic ion channels or nanopores with DNA364

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Figure 6. MicroRNA (miRNA) detection using a nanopore. a) A molecular tag is useful for generating a characteristic signal current.[48] Reproduced with permission from Ref. [48]. Copyright (2011) Nature Publishing Group. b) A theranostic system for a small-cell lung cancer. If mir-20a is detected, programmable DNA
molecules autonomously generate the DNA antisense drug for therapy of the cancer. Reproduced with permission from Ref. [50]. Copyright (2017) The American Chemical Society. c) A nanopore can estimate and generate the DNA drug without (c1) or with (c2) miR-20a in real time.[50] Reproduced with permission
from Ref. [50]. Copyright (2017) The American Chemical Society.

logic operation. These functions or technologies of such molecular machines and autonomous systems have matured sufficiently that they can operate on their own. Regarding the synthetic ion channels, early studies focused on constructing atransmembrane structure mimicking natural ion channels.
Then, the transport properties such as ion selectivity or gating
probability were investigated. Although in most cases thefunctions were carried out in a single operation, the natural
ion channels work in signal cascades, and ion transport can
induce a biological reaction. In the next step, the synthetic ionchannel will be loaded onto the interface of the molecularrobot. DNA logic operations face a similar situation. Single operations such as AND, OR, and NAND can be combined tomake sophisticated systems. Therefore, recent compelling
work has shown that combining nanopore and DNA logic operations can result in a system that can be integrated into thebody and interface with molecular robots.
Similar approaches can be found in synthetic biology andprotocell studies, in which biological technologies such as
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gene manufacturing are used. It is envisaged that by fusing
these technologies and science, in the future, microorganisms
such as molecular robots will operate in living systems for diagnosis and therapy, in other environments for assessment or
recovery, and in space for terraforming.

Acknowledgements
I wish to thank Professor Satoshi Murata of Tohoku University forproviding the beautiful illustration of a molecular robot(Figure 1). This research was supported in part by KAKENHI, “Molecular Robotics” (Grant No. 15H00803), and MEXT, Japan (Grant
No. 16H06043).

Conflict of Interest
The author declares no conflict of interest.
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Manuscript received: September 5, 2017
Accepted manuscript online: November 10, 2017
Version of record online: December 8, 2017366

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