Communication pubs.acs.org/Biomac
Covalent Cargo Loading to Molecular Shuttles via Copper-free “Click Chemistry” Susanna M. Früh,† Dirk Steuerwald,† Ulrich Simon,‡,§ and Viola Vogel*,† †
Laboratory of Applied Mechanobiology, Department of Health Sciences and Technology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland ‡ Institute of Inorganic Chemistry, RWTH Aachen, Landoltweg 1, 52074 Aachen, Germany § JARA − Fundamentals of Future Information Technologies, Germany S Supporting Information *
ABSTRACT: An important prerequisite for molecular shuttle-based functional devices is the development of adequate linker chemistries to load and transport versatile cargoes. Copper-free “click chemistry” has not been applied before to covalently load cargo onto molecular shuttles propelled by biological motors such as kinesin. Due to the high biocompatibility and bioorthogonality of the strain-promoted azide-alkyne cycloaddition, this approach has pronounced advantages compared to previous methods.
T
Scheme 1. Concept of Covalent Cargo Loading to Molecular Shuttles Using “Click Chemistry”a
he development of functional devices powered by molecular machines, which are able to transport cargo along engineered tracks, is of great interest within the interdisciplinary field of nanotechology. Today, a wide variety of molecular workhorses of either artificial or biological origin are exploited that are propelled by various schemes: synthetic approaches like catalytic motors,1 supramolecular machines,2 which are among others based on rotaxanes or catenanes and DNA walkers,3 compete with their biological counterparts, linear motor proteins (kinesin, myosin and dynein), rotary motors (F0F1-ATP synthase), or DNA motors that are responsible for crucial cellular functions.4,5 In this work, we ask how to improve the cargo loading of transport assays that are based on the well-established inverted motility assay geometry: rod-like microtubules are propelled here by the surface-adsorbed motor protein kinesin. A kinesin molecule moves along a microtubule in discrete 8 nm steps, each powered by the hydrolysis of one adenosine triphosphate (ATP) and thereby exerts a force of up to 7 pN (Scheme 1).6,7 These so-called molecular shuttles are also envisioned to serve as engines to power nanoscale hybrid devices. Such shuttles can easily be tracked by fluorescence microscopy and guided on surfaces employing chemical or topographic surface patterns as well as external stimuli such as shear stress and magnetic and electric fields.8−11 Their well-known ability to load and transport a broad range of cargoes, from molecules to virus and synthetic particles, is crucial for applications such as biosensing or sorting devices as well as molecular assembly lines. Various proof-of-concept demonstrations have been realized in a lab-on-a-chip format for this nanotransport system.5,11−14 One major task for all these possible applications and also for the promotion of artificial motor functions is the establishment of adequate linker strategies that offer fast, strong, durable, © 2012 American Chemical Society
a
The motor protein kinesin is adsorbed on a casein precoated surface. Microtubules, hollow tubes of the heterodimeric protein tubulin are actively propelled in one direction by surface-adsorbed kinesins. Molecular shuttles are integrated into a diffusion chamber made of a microscopic slide and a coverslip, which are separated via spacers. Cargo loading is achieved via the strain-promoted azide-alkyne cycloaddition of an azide-functionalized microtubule with a dibenzo cyclooctyne labeled cargo.
selective, and modular junctions between the molecular cargo and its transporter without compromising the active transport.5,15−17 Today, the most common cargo loading method in the molecular shuttle system is based on the protein−ligand Received: September 13, 2012 Revised: October 22, 2012 Published: October 29, 2012 3908
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Figure 1. (a) Aminolysis of primary amino residues on tubulin subunits of microtubules with the NHS ester of the 3.4 kDa heterobifunctional PEG linker (NHS-PEG-N3) for 1 h at room temperature in a separate tube. (b) Silver-stained SDS-PAGE gel of rhodamine and PEG-azide bifunctionalized tubulin. (A) Protein standard. (B) Rhodamine and azide bifunctionalized tubulin. The band at approximately 58 kDa corresponds to the bifunctionalized tubulin originated from the sum of 3.4 kDa PEG linker and the 55 kDa tubulin. (C) Rhodamine functionalized tubulin served as a control.
interaction of streptavidin and biotin.16,18 Among others, hydrogen bridges and van der Waals interactions make this junction one of nature’s most powerful, but noncovalent interactions.19 On the basis of this system, many cargoes such as antibodies,13,20 DNA,21,22 cyclodextrin conjugates,23 or nanoparticles such as quantum dots have been immobilized on kinesin-driven microtubules.24−26 Intrinsic to any system that allows for multifold coordination, it is inherently prone to cross-linking, such as the 4-fold coordination of biotin to streptavidin.27,28 Also, the binding strength of the cargo to the microtubule is limited to the noncovalent interaction between streptavidin and biotin whose lifetime is shortened from days to minutes when under mechanical tension.29,30 Covalent cargo immobilization strategies are thus attractive alternatives. Several covalent approaches have already been reported, but only for the prefunctionalization of microtubules that is before subjecting them into a motility assay. For this purpose, chemical cross-linking reagents such as glutaraldehyde, succinimidyl 4-hydrazidoterephthalate hydrochloride, or Nhydroxysuccinimidyl (NHS) ester-functionalized units (Table S1) have been used.31,32 These powerful reagents exclusively target the primary amino residues of any protein. They can therefore only be used to prefunctionalize microtubules. For some applications, it might also be very interesting to have multiple coexisting and specific binding chemistries to select different cargo, which is why the combination of orthogonal linker chemistries would be advantageous. In this work, we apply for the first time copper-free “click chemistry” as a compelling alternative to the existing cargo loading strategies. The most prominent “click” reaction, a copper(I)-catalyzed version of the Huisgen cycloaddition, has been introduced by Sharpless et al. as a universal coupling reaction with high efficiency and high selectivity.33,34 However, the most suitable copper(I) source in aqueous systems, namely, copper(II) in combination with a reducing agent, and copper(I) itself are not fully compatible with biological systems, and in our case was observed to aggressively destabilize microtubules (see Supporting Information, Figure S1).35,36 Therefore, we chose the biocompatible strain-promoted azidealkyne cycloaddition (SPAAC), namely, a copper-free “click”
reaction of an azide with the ring-strained dibenzo cyclooctyne (DIBO) to covalently load cargo on propelled molecular shuttles (Scheme 1).37,38 This bioorthogonal reaction is fast, selective, long-term functional, and easy to integrate into the biological transport system. Major challenges described above, particularly preventing cross-linking of molecular shuttles as well as lowering the probability of cargo loss in consequence of the highly stable covalent linkage, are addressed by the utilization of the discussed representative of copper-free “click chemistry”. Microtubules were polymerized from a mixture of unlabeled and rhodamine-labeled tubulins (purchased from Cytoskeleton) in a ratio of 7:3. The implementation of the azide moiety, which is long-term functional under aqueous conditions, was achieved using NHS ester-mediated aminolysis. The primary amino residues of the microtubules were prefunctionalized in a separate tube before the filaments were subjected to a kinesin coated surface (Figure 1a). The polymerization of microtubules from the tubulin dimer was conducted before functionalization to ensure structurally intact and motile microtubules, while accepting that a considerable fraction of lysine residues might be inaccessible due to geometric constraints in the microtubule itself. For prefunctionalization, a 3.4 kDa heterobifunctional poly(ethylene glycol) (PEG) cross-linker was chosen, that carried the reactive NHS ester as well as the azide moiety. With the help of the PEG spacer, we allowed the functional group on the microtubule to be more accessible for potential cargo molecules due to steric reasons. Furthermore, the PEG linker allowed control for the success of the prefunctionalization by gel electrophoresis: functionalized tubulin possesses an additional 3.4 kDa molecular weight than nonfunctionalized tubulin. Using silver-stained reducing sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and successive silver-staining, we could distinguish between functionalized and nonfunctionalized tubulin (Figure 1b). The prefunctionalized microtubules were subjected to kinesin-coated surfaces of a standard inverted motility assay and showed full motility with an average gliding velocity of 0.59 ± 0.04 μm s−1, which is in good agreement with the literature.32 The subsequent “click” reaction was conducted under the standard physiological buffer conditions for inverted motility 3909
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Figure 2. (a) Confocal fluorescence microscopy images of azide-functionalized molecular shuttles after copper-free “click” reaction with the DIBOlabeled Alexa Fluor 488 dye (30 min at room temperature). Right column: Colocalization of microtubules and cargo confirms the successful loading via SPAAC. Left column: After incubating rhodamine-labeled microtubules without azide functionality for 30 min with DIBO-functionalized cargo, no reaction occurred. (b) Confocal fluorescence microscopy images showing motile microtubules over a period of 15 s carrying the Alexa Fluor 488 dye. Scale bar corresponds to 10 μm.
Covalent cargo loading by SPAAC is a versatile method and can easily be implemented into the standard solutions and conditions of microtubule gliding assays. Spontaneous cargo loss, which hampered efficient long-range transport in many of the previous proof-of-principle experiments, is effectively excluded by the covalent bond of the formed triazole.22,39,40 Cross-linking of microtubules is intrinsically precluded due to the highly selective and bioorthogonal copper-free “click” reaction. Consequently, no filament bundles or spools could be found, which had previously been observed when linking streptavidin to biotinylated microtubules.27 Furthermore, the negligible molecular weight of the formed triazole bond together with the PEG spacer is sterically favorable over protein−ligand interactions, mediated, for example, by the 60 kDa protein streptavidin that can hamper the transport function.15 In summary, we established the copper-free “click chemistry” approach represented by the SPAAC that has been applied to covalently load molecular cargo on motor-driven shuttles without compromising the fragile functions of the microtubules. The implementation is straightforward and offers new perspectives for loading of biological and synthetic cargoes. Since both functional groups, the azide and the DIBO, are already commercially available with many chemical functionalizations, we expect that a broad range of cargo molecules can easily be coupled to molecular shuttles including biomolecules such proteins or DNA,41 biological entities such as liposomes, viruses or organelles, and nanotechnological building blocks such as nanoparticles,42 nanocontainers,43 quantum dots, and many more.44 Regarding the various synthetic approaches such as catalytic motors and their emerging cargo loading ability, this same strategy could be a useful tool for specific cargo loading
assays. We injected an Alexa Fluor 488 dye carrying the DIBO functionality as a model cargo. After an incubation time of 30 min, which was estimated to allow for a full conversion (see Supporting Information, Figure S2), the dye was located exclusively to the microtubules. This was confirmed by the colocalized fluorescence signals of rhodamine and Alexa Fluor 488 (Figure 2a). Nonspecific binding of the dye was ruled out by 30 min incubation of nonfunctionalized microtubules with the model cargo, which showed no signal in the Alexa Fluor 488 channel. Furthermore, the cargo loading reaction did not influence the microtubule motility (Figure 2b). The analysis of microtubule length revealed that the molecular shuttles did not depolymerize during and after cargo loading via SPAAC. The average length was calculated to be 5.3 ± 1.7 μm throughout the complete duration of the experiments. Additionally, the molecular shuttle velocity did not show significant changes during or after SPAAC-mediated cargo loading that could be ascribed to negative effects on protein function (Table 1). Table 1. Average Microtubule Length and Gliding Velocity before, during, and after Cargo Loading via SPAAC at a Constant ATP Concentration of 10 μM reaction time
0 mina
10 minb
40 minc
microtubule length/μm microtubule velocity/μm s−1
5.3 ± 1.9 0.59 ± 0.04
5.2 ± 1.7 0.64 ± 0.05
5.4 ± 1.5 0.70 ± 0.06
a
Corresponds to a time point before the addition of DIBO functionalized Alexa Fluor 488. bCorresponds to a time point during the SPAAC-mediated cargo loading. cCorresponds to a time point after the cargo loading via SPAAC. 3910
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onto these molecular machines as well. Copper-free “click chemistry” can easily be integrated into other also nonbiological systems45 and thereby offers a new platform for cargo loading and designing nanoscale biosensors, sorting devices, as well as molecular assembly lines.
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ASSOCIATED CONTENT
S Supporting Information *
Background information. Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Prof. Henry Hess for providing the kinesin. The financial support from ETH Zürich is gratefully acknowledged. REFERENCES
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