DOI:10.1002/cphc.201700982 Minireviews Synthetic Ion Channels and DNA Logic Gates as Components of Molecular Robots Ryuji Kawano*[a] Amolecular robot is anext-generation biochemical machine sensorsare useful components for molecular robots with that imitates the actions of microorganisms. It is made of bio- bodies consisting of alipid bilayer because they enablethe in- materials such as DNA, proteins,and lipids. Three prerequisites terface between the inside and outside of the molecular robot have been proposedfor the construction of such arobot:sen- to functionasgates. After the signal molecules arrive inside sors, intelligence, and actuators. This Minireviewfocuseson the molecular robot, they can operate DNA logic gates, which recent research on synthetic ion channels and DNA computing perform computations. These functions will be integrated into technologies, which are viewed as potential candidate compo- the intelligence and sensor sections of molecularrobots. Soon, nents of molecularrobots. Synthetic ion channels, which are these molecular machines will be able to be assembled to op- embedded in artificial cell membranes (lipid bilayers), sense erate as amass microrobot and play an active role in environ- ambient ions or chemicals and import them. These artificial mental monitoring and in vivo diagnosis or therapy. 1. Molecular Robots and the Lipid Bilayer Platform Molecular robots have recently emerged based on biomole- source by chemotaxis. These accomplished functions are inte- cules andbiochemical processes. Approximately30years ago, grated in amicron-sized body surrounded with abilayer lipid aself-constructing machine, aso-called “assembler,” was origi- membrane (BLM). Sato et al. reported the development of a nally proposed by Drexler.[1] Based on the idea of an assembler, sophisticated molecularrobot prototype in 2017.[9] Their devel- molecular machinesthat operate autonomously have been de- oped amoeba-type robot has light-induced DNA clutches for velopedusing DNA or RNA. For example, DNA walkers move sensors and kinesin-microtubule proteins as actuators, all inte- autonomously,onthe basis of energy supplied from the hy- grated in acell-sized liposome. Light irradiation acts as atrig- bridization of fuel oligonucleotides, from one binding site to ger for the releaseofthe signal molecules and disengagement another on aDNA-modified surface.[2,3] Rothemund has also of the DNA clutches to change the shape of the liposome. proposed amethodbywhich DNA molecules can be folded The fabrication process used for existing mechanical robots into any desired two-dimensional shape to make “DNA origa- is viewed as ablueprint for the manufacture of these molecu- mi.”[4] In the field of synthetic chemistry,nanosized machines lar robots. In the case of humanoid robots, the arms and legs such as motors and ratchets have been developed based on are manufactured individually andthen assembled. Similarly, organic or supramolecular chemistry.[5–7] Thiswas viewed as individual fabrication would be astraightforward process in ground-breaking technology and the pioneers have since been the manufacturing of molecularrobots. Hence, aprototyping honored with the Nobel Prize in Chemistry 2016. factory is required, as well as an industrial manufacturing pro- Inspiredbythe idea of amolecular assembler,molecular ro- cess. botics, whichinvolvesconstruction with amuch higherdimen- In this Minireview,Ifocusonmanufacturing the body of the sion of assembly,was proposed in 2014.[8] Molecularrobots are molecular robot using BLM with membrane receptors composed of sensors,calculators, andactuators that are all im- (Figure 1). BLM is the ideal materialfor the body of molecular plementedinliposomes or hydrogels. White blood cells, the robots because it is naturallybiocompatible and can host re- most imageable example, senses the chemical signals secreted ceptor proteins. In addition, physicaldynamics such as mem- from atarget bacterium, calculates the direction and length brane fusion and endo- or exocytosis, such as molecular between the target and itself, and moves towardthe signal uptake processes, are useful for the interfaceofthe robots. We previously developed ahigh-throughput planar BLM (pBLM) [a] Dr.R.Kawano system that will be apowerful tool for the manufacturing of [10–12] Department of Biotechnologyand LifeScience the lipid body of molecular robots. ApBLM is generally Tokyo University of Agricultureand Technology(TUAT) used as the ion current measurement of an ion channel or 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588 (Japan) pore-forming proteins. Because the reproducibility and stability E-mail:[email protected] of pBLMs are conventionally insufficient, variousmethods have The ORCID identification number for the authorofthis articlecan be found under: https://doi.org/10.1002/cphc.201700982. been proposed to overcome this, primarily using microfluidic [13] An invited contribution to aSpecialIssue on Reactions in Confined technology. The most promising method is the dropletcon- Spaces tact method,[14,15] in which two microdroplets surrounding the ChemPhysChem 2018, 19,359 –366 359 2018 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Minireviews DNA computing technology with nanopore proteins will be able to connect the sensorand intelligence functions. 2. Synthetic Ion Channels and Transport Control at the Lipid Bilayer Membrane Receptor proteins sense specific molecules in the cell mem- brane.When the ligand molecule binds to areceptor,the re- ceptor senses the binding and produces asignal that is trans- mitteddownstream to control ion transport through ion chan- nels, as it cascades.Furthermore, pore-formingproteins play a key role in the transportation of molecules with the gradient of membrane potential or the substrate concentration.Artificial ion channels or pores have been created using synthetic chemistry to mimic the structure or functionofnatural pro- Figure 1. Conceptual illustration of amolecular robot. The body consists of [19,20] alipid bilayer with synthetic ion channels as the gate. DNA computing archi- teins. In the last three decades, synthetic chemists have tecture is integratedinside the robot as the intelligence. (This illustration is proposed various compounds that actively exhibit ion channel used courtesy of Professor S. Murata of TohokuUniversity). structures and functions.[21] As part of the design,chemists try to add functions such as pore size, ion or substitute selectivity, lipid monolayer are brought into contact and astable and re- and voltage or ligand gating.Inthe early studies, researchers producible pBLM is formed at the droplet interface,asthe imitated natural compounds, such as valinomycin or gramici- droplet–interface bilayer.This methodissimple and rapid, and din, forthe structuralframework of synthetic channels (Fig- the formed pBLM is extremely stable. Based on this method, ure 2a). Valinomycinisamacrocyclic polypeptideused in the severalhigh-throughput pBLM formation methods have been transportofpotassium, as an antimicrobial peptide. Gramicidin proposed and appliedtolarge-scale measurement of ion chan- is also apolypeptide and forms a b-helix structure in the lipid nels or nanopore measurements.[16–18] monolayer;[22] within the bilayer,two gramicidin molecules In the following section, two recent efforts are introduced form an end-to-end dimer,which in turn forms atransmem- that attempted 1) to use asynthetic ion channel as the artifi- brane structure in the bilayer.These macrocyclic or dimer cial receptor protein, and 2) autonomous sensing and calcula- structures have been modeledfor the design of synthetic tion using DNA computing with nanopore technologyusing a channels, and chemistssynthesized the mimicking structure high-throughput pBLM system. Synthetic channels are poten- using synthetic peptides or macromolecules. More recently, tial candidates for the sensor sections of molecular robots. other functional materials such as DNA origami and carbon nanotubes have been proposed as potential candidates for ar- tificial ion channels.[19,23, 24] One of the most soughtafter properties is ion selectivity for Ryuji Kawano was born in Oita, Japan + + 2+ Na ,K ,Ca ,ClÀ ,and other ions. Most studies have focused in 1976. He received his Ph.D. in 2005 on synthesis of the channel structure in the lipid membrane, from Yokohama National University and assessed the ion selectivity.Among them,the first report under the supervision of Prof. Ma- of aselective channel was for K+ ions.[25] In nature, potassium- sayoshi Watanabe, working on dye- channel proteins use aselective filter that has five amino acids, sensitized solar cells using ionic liquid TVGYG, within each of the four subunits.The hydrated K+ ion electrolytes and analyzing the charge is dehydrated by interacting with these motifs, and then transport mechanism using electro- passesthrough the filter pore with high ion selectivity chemistry.Subsequently,hespent ap- (Figure 2b).[26] Conversely,inthe synthetic channels, the proximately three years in Prof. K+-selective filter is composed of an aromatic ring (primarily Henry S. White’slaboratory at the Uni- formed by four tyrosine residues)that provides aweak electric versity of Utah as apostdoctoral re- field, allowing complete dehydration of K+ ions, but not of searcher,where he conducted research on nanopore measure- Na+ ions. ments with glass materials. He then joined Prof. Shoji Takeuchi’s In this system, the p electrons of the aromatic rings contrib- group at the Kanagawa Academy of Science and