Commentary pubs.acs.org/accounts
Swallowing a Surgeon: Toward Clinical Nanorobots Published as part of the Accounts of Chemical Research special issue “Holy Grails in Chemistry”. Seunghyun Sim† and Takuzo Aida*,†,‡ †
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡ RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ABSTRACT: In this Account, “a step toward clinical nanorobots” is proposed as one of the Holy Grails in chemistry, which could lead to a great leap in the field of biomedicines when accomplished. We review our preliminary contributions to this challenge by engineering chaperonin protein GroEL to generate de novo structures and functions.
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INTRODUCTION It would be interesting in surgery if you could swallow the surgeon. You put the mechanical surgeon inside the blood vessel and it goes into the heart and “looks” around... How do we make such a tiny mechanism? I leave that to you.
For understanding the current situation, we note remarkable progress in the last decades in the development of synthetic molecular machines.4−17 The pioneers of such molecular machines acknowledged by the 2016 Nobel prize in chemistry; Sauvage, Stoddart, and Feringa, designed particular molecules including interlocked molecular systems to respond to external stimuli for exerting directional mechanical motions.4−10 This research field contributed remarkably to the fundamental understanding of how molecular motions are controlled externally from the macroscopic world. Nevertheless, we must admit that even the frontier of artificial molecular machinery, though approaching the level of what synthetic chemistry can do ultimately, is still far from biological molecular machines in terms of the complexity, sophistication, and scope. For example, despite some limited exceptions,14−17 these systems generate a simple output in response to a single or double input and are not capable of processing multiple data inputs by an intricate logic gate mechanism. It should also be noted that most artificial robotic systems cannot sense endogenous signals, although some hydrogels were designed to swell and shrink in response to certain substances of biological importance such as glucose and Ca2+.18−20 Thus, numerous challenges still exist for reducing the large gap between synthetic and biological molecular machines. Biomolecular nanorobots, evolved by nature based on proteins, achieve a variety of biologically important functions with high fidelity using concerted mechanical motions driven by endogenous signals such as adenosine triphosphate (ATP), guanosine triphosphate (GTP), and nucleic acids.21,31 Such omnipotent biological machines are extraordinarily sophisticated and might act as a template for the design principles of clinical
R. Feynman1
The first and most famous person to introduce the concept of “swallowing a surgeon” was Richard Feynman. In his famous speech “There’s plenty of room at the bottom”, he described an idea of infinitesimal nanorobots that are able to precisely and autonomously cure a disease. Even after numerous breakthroughs in the field of medicine, a noninvasive cure of diseases by nanoscopic robots is still a virtual goal, and the realization of this ambition would arguably deserve the title of a Holy Grail. What are the prerequisites for such clinical robots? Clinical robots have to be small enough not to physically deteriorate living tissues upon gaining entry to our body for operations. At the same time, these robots have to be large enough to accommodate multiple sensing units for endogenous as well as exogenous signals. The sensing units should be designed to work cooperatively just like logic gates2,3 in order for the robots to wisely judge every second which programmed task they need to implement. Hence, in most cases, such clinical robots may comprise multiple nanoscopic devices whose job purposes are different from one another. Discrete assemblies that carry only a reporter function for endogenous signals might be called diagnostic clinical robots. However, robots that really deserve the title of a Holy Grail would have to additionally carry mechanical drive units that allow such robots to catch a hold of the target proteins or even arrest viruses to disable or modify their intrinsic functions. There are a multitude of issues to argue about how state-of-the-art clinical robots should be 50 years from now. © 2017 American Chemical Society
Received: September 30, 2016 Published: March 21, 2017 492
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co-workers further demonstrated that forced reverse-rotation of this rotor could lead to the synthesis of ATP.28 Recently, ClpX protein, originally serving as an assembling unit of Clp protease in nature, was harnessed as a membrane translocator for a protein guest through the α-hemolysin pore.29 There has also been an attempt to utilize a small chemical moiety to add an anomalous function to the existing protein machinery: Nolte and co-workers attached a manganese porphyrin to the DNA binding clamp protein gp45 to construct a biohybrid DNA oxygenating catalyst.30 Pioneering examples to generate de novo functions of biomolecular machines are summarized in Figure 2. However, none of such milestone examples described above has stood in the way of the Holy Grail suggested herein. From the section below, we summarize years of our efforts in engineering GroEL machinery as a preliminary step toward the functional clinical nanorobots.
nanorobots. Their highly complicated and precise mechanical motions, processing the data by the combination of allosteric effects, are thus far beyond the mechanical motions realized by artificial robotic systems to date. Such fascinating capabilities enchanted the scientific society and led us to the questions regarding what they actually “look” like, which brought about great advances in structural characterization of particular proteins. By site-specific hybridization with artificial molecular systems, one may envision that such biomolecular nanorobots could be elaborated not only genetically but also chemically for the realm of clinical nanorobots. Tasks that clinical nanorobots are designed to perform need not to be confined to the intrinsic roles of their constituent units but could rather be repurposed to perform new functions, such as incapacitating viruses or cancer cells. We believe that this research direction is indeed one of the virtual scientific goals that can be shared by all human beings for the next 50 years (Figure 1).
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MOLECULAR ENGINEERING OF CHAPERONIN GroEL As one of the first steps toward the grand challenge of developing clinical nanorobots, we started a new project in early 2000 to chemically explore a unique protein assembly, GroEL, from Escherichia coli. GroEL is a chaperonin that serves as a machine to facilitate the refolding of denatured proteins.31 Its cylindrical hollow structure with a double-decker geometry, comprising two cyclic heptamers of an identical protein, undergoes chemomechanical opening and closing motions fueled by ATP upon binding and its subsequent hydrolysis into adenosin diphosphate (ADP). GroEL facilitates refolding of denatured proteins by sequestering them inside its cavity (Figure 3a). As summarized in the following sections, we have so far been motivated to test a variety of ideas to incorporate GroEL in the design of nanorobotic machinery with genetic and chemical modifications. Initially, we investigated whether guest-capture and release mechanisms may work for nonbiological guests. If such ATP-fueled events are restricted to proteinaceous guests, our attempt would not have been successful. As reported in 2003, we chose CdS quantum dots (QDs) with a diameter of 2−3 nm as our first nonnatural guest.32 When mixed with GroEL, QDs are incorporated into the GroEL cavity and their coagulation is inhibited. Analogous to the biological function of GroEL, addition of ATP with a hydrolysis cofactor Mg2+ to this GroEL/QD complex resulted in the release of QDs, demonstrating that the mechanical motion of GroEL fueled by ATP is applicable to the reversible trap and release of nonbiological guests as well as proteinaceous guests (Figure 3b). This finding motivated us to move further to the decoration of the protein shell with synthetic moieties. The next goal we set was to produce a logic gate combining external stimuli with the ATP-fueled conformational change of this protein assembly.33 Thanks to the GroEL structure, which is tolerant enough to bare multiple mutations without altering its native shape, several cysteine mutants have been produced by converting all the naturally occurring cysteine residues to alanine for the purpose of realizing site-specific cysteine mutations. The first cysteine mutant harnessed was CA-R231C, having one cysteine residue at the apical domain of each constituent monomer, thus bearing 7 cysteine units near the substrate-binding gate on each side of the cylinder. These residues could be modified chemically with an azobenzene moiety by maleimide/thiol bio-orthogonal conjugation, resulting in a protein that showed a binary response not only to ATP but also to light (GroELazo). We found that the application of both ATP and UV light turns on an “And” logic gate based on GroELazo and actively refolds denatured green fluorescence protein (GFP, Figure 3c). Through these investigations, we learned that the GroEL systems (1) can also
Figure 1. Schematic image to illustrate the Holy Grail: Proteinaceous clinical nanorobot.
Well-defined protein machines include (1) motor proteins such as kinesin, dynein, and myosin, (2) membrane protein pumps such as F0/F1-ATPase and various ion channels, (3) cofactors for protein folding such as ClpX/P and GroEL/ES, and (4) units that are involved in the process of central dogma such as DNA/RNA polymerase, ribosomes, and their cofactors. Some of these machineries have been molecularly engineered in recent years from their native structures to introduce de novo functions (Figure 2).21−38 For example, in recent mutation studies on motor proteins by Bryant and co-workers, several strategies have been demonstrated to control the movements of these proteins.23,24 Recombination of structural modules has made it possible to generate a series of novel proteins that respond to external stimuli and change the direction of the power stroke accordingly. Directional movements of motor proteins were harnessed in various cargo transports, including the applicationoriented demonstration done by Hess and co-workers to build up a dust-biosensor by kinesin motors.25 Manipulating natural rotor F1-ATPase to rotate artificial molecules such as actin fibers and metallic nanorods was reported in early 2000.26,27 Noji and 493
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Figure 2. Summary of the recent progress in implementing de novo functions to biological proteinaceous machines.21−38
Figure 3. Molecular engineering of chaperonin GroEL. (a) Crystal structures of GroEL before (left; PDB ID 2NWC) and after (right, PDB ID 4AAS) the ATP-induced structural transformation. (b) Schematic representation of the capture and ATP-induced release of a nonbiological guest, such as a CdS quantum dot, by GroEL.32 (c) An azobenzene-appended GroEL mutant (CA-R231C) and its function as an “AND” logic gate responsive to ATP and UV light.33
which has double the cysteine residues on its apical domain (2 per monomer unit, 14 overall). By using the increased number of cysteine moieties for anchoring, we attached merocyanine (MC) moieties to the apical domains of the GroEL mutant in a manner analogous to that reported in 2006.34 The resulting GroELMC self-assembles one-dimensionally into a nanotube structure upon the addition of divalent metal ions such as Mg2+ to enable multiple merocyanine−metal ion coordination, which
handle nonbiological guests while maintaining their intrinsic functions and (2) allow site-selective cysteine mutations to modify proteins with a specific chemical moiety of our choice.
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DE NOVO PROTEIN NANOTUBES USING CHAPERONIN PROTEINS TOWARD “CLINICAL NANOROBOTS” For the purpose of engineering GroEL with more functional groups, we employed a novel GroEL mutant CA-K311C/L314C, 494
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Figure 4. De novo nanotube structure and functions based on GroEL machinery. (a) Merocyanine (MC)-modified GroEL mutant (CA-K311C/L314C) that forms a de novo nanotube structure by coordination of Mg2+ with MC.34 (b) Reversible isomerization between MC and spiropyran (SP) by irradiation with UV/visible light resulting in the polymerization and depolymerization of the nanotube.35 (c) GroEL nanotube acting as an ATPresponsive drug carrier.37 (i) Model prodrug, a cyanine dye attached via an ester bond to the denatured lactoalbumin scaffold, is encapsulated by GroELMC. (ii) The resulting GroELMC/prodrug complex is polymerized to form a nanotubular assembly via Mg2+. (iii) The prodrug-containing nanotube, when taken up into the cytosol, dissociates into short-chain oligomers by the action of high-concentration ATP. (iv) The exposed ester linkage of the prodrug is then cleaved off by the cellular esterase, resulting in the formation of cyanine dye CYred, which luminesces at 611 nm. (d) Formation of an anisotropic superparamagnetic nanoparticle (SNP) array inside the GroEL nanotube and its magnetic-field responsive reversible assembling behavior.36 (e) Rationally designed end-capper to the GroEL nanotube that can dimerize by itself and modulate the nanotube length.38
bundles by a magnetically enhanced lateral interaction. This interesting behavior is reversible and reminiscent of the distinct assembling behaviors of certain biological protein fibers in the cell. Overall, the GroEL nanotube is an anomalous protein assembly, whose 1D and 3D assembling behaviors can be manipulated from the macroscopic world. Whether this nanotube, formed by consecutive connections of the GroEL machinery, would still be responsive to ATP was our central question. Upon treatment with ATP, the nanotube dissociates into its short-chain oligomers, including the monomer (Figure 4c).37 The bound ATP induces a conformational change of the constituent GroELMC units, and this generates chemomechanical motions in the nanotube that are large enough to disrupt the 1D assembled structure. A highly twisted, wavy morphology of the short chain oligomers after ATP addition further supported this claim. We encapsulated a model prodrug, a cyanine dye attached via an enzymatically cleavable ester linkage to an irreversibly denatured lactoalbumin scaffold, in the GroELMC monomer. This model prodrug is designed to change its luminescence if the connecting ester linkage is cleaved. The guest-containing monomer GroELMC can be polymerized via Mg2+ into a GroEL nanotube carrying the model prodrug in its interior. After postmodification at its surface with a boronic ester motif, this guest-containing nanotube was subsequently
was evidenced by the absorption spectra and EDTA-induced dissociation. This is the first report of a de novo protein nanotube (Figure 4a).34 Notably, the nanotube has an exceptionally high thermodynamic stability, so that it is stable under size exclusion chromatography conditions just like covalent polymers. Nevertheless, when exposed to visible light (λ > 400 nm) to isomerize the coordinating merocyanine units into spiropyran having no metal coordination ability, the nanotube is cut into short tubular fragments (Figure 4b).35 Thus, this 1D protein assembly is highly stable thermodynamically due to multivalent connection of the constituents but turns dynamic by the exposure to external stimuli such as light. In addition to the modulation of this 1D polymeric assembly, we were seeking a method to control its 3D lateral assembly. It has come to our mind that the topology of this nanotube is indeed distinctive from other natural or artificial nanotubes: constituent GroELMC in reality has two discrete interior spaces separated by a small wall between the two halves. Harnessing this unique structural feature of the GroEL nanotube, together with its capturing ability of guests including artificial ones, we constructed a very long GroEL nanotube containing superparamagnetic nanoparticles (SNP). This can be regarded as a long 1D array of SNPs encapsulated by the GroEL nanotube as a jacket (Figure 4d).36 This 1D SNP array is magnetically responsive; Upon applying a magnetic field, it self-assembles into 495
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perfect and ultrafast molecular recognition and helps us imagine what virtual protein nanorobots we should pursue beyond the scope of clinical nanorobots. In the future, we hope to see expansion and great progress of the related fields.
taken up into HeLa cells and broken up into short chain oligomers by the action of intracellular ATP. As a result, the model prodrug is exposed to the cytoplasm, where esterase acts to cleave off the ester linker, thereby releasing the cyanine dye from the GroEL cavity. Of particular importance, the ATP concentrations in intracellular matrices are nearly 10 000 times greater than those in extracellular matrices. This gives us a great motivation to develop this ATP-responsive nanotubular protein machinery toward real application as a cancer-specific drug carrier. Fortunately, biodistribution tests using tumor-bearing mice reveal that this robotic nanotubular carrier is preferentially accumulated in tumor tissues over other tissues, except liver. For further elaboration of this robotic nanotubular carrier, it is necessary to clarify how its length affects the cellular uptake efficiency and circulation behavior. Recently, we reported a new GroEL mutant SRMC, a half-cut version of GroELMC exploited by applying a single ring mutation to the cysteine mutant CA-K311C/L314C.38 In the Mg2+-mediated supramolecular polymerization of GroELMC, half-cut version SRMC successfully serves as an end-capper for the GroEL nanotube, thereby controlling the average length of the resultant nanotube (Figure 4e). This achievement allowed us to investigate the cellular uptake behavior of GroEL nanotubes of varying lengths. From this study, we found that the length of the GroEL nanotube significantly affects the cellular uptake efficiency, and nanotubes longer than 100 nm are less likely to be taken up into cells.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Takuzo Aida: 0000-0002-0002-8017 Notes
The authors declare no competing financial interest.
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REFERENCES
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PERSPECTIVES In pursuit of the Holy Grail, we have continuously expanded the design space of GroEL and its polymer since 2003. Based upon the knowledge accumulated so far, we would like to take another plunge to push this study forward to clinical applications. For instance, chemically and biologically unstable drugs such as siRNA could become attractive targets. Nevertheless, for de novo design of proteinaceous nanorobots, we need many more tools than we have now. One of them would be a novel computational approach. Recent advances in computational understanding of protein assemblies are remarkable:39−41 Baker and his colleagues designed de novo protein complexes bearing multiple symmetry axes by the state-of-art computational algorithms. In fact, such sophisticated protein complexes were synthesized and revealed to bear the expected structures. Extensive application of this state-of-the-art computational technology to the research field of protein machineries may provide the great potential to finely tune the mechanical motions of biological machines and to realize seamless conjugation of such intrinsic functions with other functions added by genetic and chemical mutations. In relation to the chemical mutation, noteworthy are recent achievements in the development of bio-orthogonal click reactions that allow site-specific modifications of target proteins with non-natural chemical functionalities.42−45 Having these powerful tools and extensive collaborations, one may achieve anomalous mechanical motions that are not intrinsically designed to operate. Needless to say, an even more challenging Holy Grail along this line is the ribosome. Ribosomes synthesize proteins out of 20 different amino acid reservoirs by correctly reading out the information written in the target mRNA template. Ribosomal protein synthesis controls the polymer length, stereochemistry, and sequence all at once, which is far superior to the artificial polymer synthesis. A harmonious, sophisticated, and prompt motion of this gigantic protein machinery in response to its surrounding environment is a shining real world example of 496
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