Protein Nanotube Selectively Cleavable with DNA: Supramolecular

Dec 11, 2017 - Here, we report molecular chaperone GroELs that carry, at their apical domains, multiple DNA strands (ideally 28 DNA strands in total) ...
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Protein Nanotube Selectively Cleavable with DNA: Supramolecular Polymerization of "DNA-Appended Molecular Chaperones" Daiki Kashiwagi, Seunghyun Sim, Tatsuya Niwa, Hideki Taguchi, and Takuzo Aida J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 11, 2017

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Protein Nanotube Selectively Cleavable with DNA: Supramolecular Polymerization of “DNA-Appended Molecular Chaperones” Daiki Kashiwagi,† Seunghyun Sim,*,† Tatsuya Niwa,§ Hideki Taguchi,§ 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 §

Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, 226-8503, Japan

‡

RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Supporting Information Placeholder ABSTRACT: Here we report molecular chaperone GroELs that carry, at their apical domains, multiple DNA strands (ideally 28 DNA strands in total) with defined oligonucleotide (nt) sequences. This design strategy allows for the preparation of GroEL10a and GroEL10b carrying 10-nt DNA strands of 10a and 10b with complementary sequences, respectively, at their apical domains (Table 1). One-dimensional coassembly of these GroELs is possible to form protein nanotube NT10a/10b with an anomalous thermodynamic stability due to the exceptionally large multivalency for the coassembly. Likewise, comparably stable nanotube NT15c/10d was obtained even when the apical-domain DNA strands (15c and 10d) were partially complementary to one another. Nevertheless, in sharp contrast with NT10a/10b, NT15c/10d, when incubated with DNA 15d, dissociates rapidly and completely because 15d preferentially hybridizes with the DNA strands of 15c in NT15c/10d by displacing those of 10d, to afford a mixture of GroEL15c/15d and GroEL10d. Even in the presence of NT10c/10d, 15d cleaved off NT15c/10d selectively, indicating the potential utility of NTs for targeted delivery.

Supramolecular polymerization offers a myriad of functions emerging from various 1D nanoscale structures.1 Amongst them, nanotubes are interesting, because they can transport multiple guest molecules simultaneously.2 Increasing attention has been paid to nanotubes that can reversibly fragment into short pieces in response to external stimuli. Especially, as represented by microtubules that are responsive to GTP,3a nanotubes that dissociate into their monomers in response to biological stimuli are highly attractive in view of their wide potential applications. However, such nanotubes are still very rare.3b, 3c Here, we report a new class of protein nanotubes that can break up completely in response to externally added DNAs in a sequence-specific manner. Self-assembly via the hybridization of complementary DNA strands provides a powerful

tool to achieve highly sophisticated nanostructures.4 In the present work, we developed the first molecular chaperone GroELs that covalently carry, at each of their apical domains, DNA strands with defined oligonucleotide (nt) sequences (Table 1). These modified GroELs, under appropriate conditions, one-dimensionally (1D) coassemble by the hybridization of complementary DNA strands in their apical do-

Figure 1. (a) Schematic illustration of the hybridization and displacement of fully and/or partially complementary DNA strands attached to GroELs: GroELCys and a 15-nt DNA appended with a fluorophore (15FL) are conjugated to yield GroEL15FL [I], which is mixed with 20-nt DNA 20Q appended with a quencher moiety to give GroEL15FL/20Q [II]. To this mixture is added 20-nt DNA 20D, which is complementary to 20Q, thereby liberating GroEL15FL with a release of 20Q/20D [III]. (b) Native-PAGE profiles of GroELCys, [I], [II], and [III], from the left. (c) Fluorescence intensities of the solutions of [I], [II], and [III] (λext = 470 nm, λem = 515 nm). These experiments were repeated 3 times independently.

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Table 1. List of complementary DNA pairs employed and their melting temperatures measured under experimental conditions a

a

In 50 mM Tris-HCl buffer containing 100 mM KCl (pH 7.6). FL: 6Carboxyfluorescein, Q: Black Hole Quencher®–1

mains, ideally in a [14 + 14] multivalent manner, affording thermodynamically stable nanotubes. By using partially complementary DNA strands for the connection of GroELs, a nanotube that contains single-stranded toehold regions can be obtained. This nanotube is likewise stable thermodynamically but can dissociate into its constituent GroEL monomers by the action of a fully complementary DNA strand.

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In 2009, we developed a merocyanine-appended GroEL derivative using a GroEL mutant CA–K311C/L314C (GroELCys) bearing cysteine residues at its apical domains.5 GroELCys was successfully expressed from E. coli, according to previous reports (see Supplementary Methods and Figure S1).5c, 5d In order to covalently attach 10-nt DNA 10a to its apical domains, 5 equivalents of maleimide-functionalized 10a were mixed with GroELCys (0.5 µM, Tris-HCl 50 mM, KCl 100 mM, MgCl2 50 mM, pH 7.6; for details see Supplementary Methods). After 2 h at 37 °C, the reaction mixture was subjected to polyacrylamide gel electrophoresis (PAGE; stained with Coomassie brilliant blue (CBB) and SYBR® Green Ⅱ) to confirm the formation of GroEL10a.6 In SDS-denaturing PAGE, a clear band shift toward the higher molecular weight region (Figure S2) was observed, together with the band visualization by staining with SYBR® Green Ⅱ (Figure S2). The band intensity area map, generated from the PAGE profiles (see Supplementary Methods and Figure S2) using the imageJ software indicated that only ~5% of the GroELCys subunits remained unmodified with 10a. In transmission electron microscopy (TEM), GroEL10a showed a ring-shaped top view with 4 stripes on its side view, typical of the GroEL family (Figure S4). The hydrodynamic diameter of GroEL10a, as estimated by dynamic light scattering (DLS), was 17.3 nm, which is close to, but as expected, slightly larger than the hydrodynamic diameter (15.7 nm) of intact GroELCys (Figure S5). We also prepared GroELs with DNA strands of different sequences as listed in Table 1. To investigate whether the DNA hybridization strategy allows for noncovalent functionalization of the apical domains of GroEL, 15FL-appended fluorescent GroEL (GroEL15FL) was incubated with DNA 20Q appended with a quenching unit at [20Q]/[15FL in GroEL15FL] = 4 for 2 h at 25 °C (Figure 1a). This reaction mixture showed a band shift from [I] to [II] in native-PAGE (Figure 1b), together with a 90%fluorescence quenching from [I] to [II] in Figure 1c. To this solution was added 20-nt DNA 20D at [20D]/[15FL in GroEL15FL/20Q] = 8, which is fully complementary to 20Q, and the mixture was subsequently incubated for 16 h at 25 °C. As shown in Figure 1b, a band shift from [II] to [III] took place in native-PAGE, along with an 89%-fluorescence recovery from [II] to [III] in Figure 1c. Therefore, one can easily attach functional groups noncovalently to the apical domains of GroEL and detach them later, if necessary, by the DNA hybridization strategy (Figure 1a).

Figure 2. (a) Schematic illustration of the 1D coassembly (noncovalent alternating copolymerization) of GroEL10a and GroEL10b to yield NT10a/10b. (b) TEM image (inset; scale bar = 20 nm) of an air-dried sample of NT10a/10b stained with uranyl acetate. (c) Size-exclusion chromatography (SEC) and (d) dynamic light scattering (DLS) profiles of GroEL10a (green), GroEL10b (orange), and NT10a/10b (black). SEC traces were monitored by a UV detector (λ = 280 nm).

One of our major challenges was to investigate whether the GroEL nanotube can be constructed by the DNA hybridization strategy described above. For this purpose, we prepared GroEL10a and GroEL10b, whose apical-domain 10-nt DNA strands (10a and 10b, respectively) are complementary to one another, and unambiguously characterized them by TEM (Figure S4), SEC (Figure 2c, green and orange curves), SDS-PAGE (Figure S6), and DLS (Figure 2d). These DNA strands were chosen such that the melting tem-

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perature Tm of their hybrid (39 °C) is lower than the thermal denaturation temperatures of GroEL mutants (60–70 °C, Figure S7). For their coassembly, 50 mM Tris-HCl buffer solutions of GroEL10a and GroEL10b, both containing 100 mM KCl (pH 7.6), were gently heated for 5 min at 50 °C to avoid kinetic aggregation and then mixed together equimolarly. The resulting solution was slowly cooled to 20 °C at a rate of 0.1 °C/s, whereupon protein nanotube NT10a/10b, as observed by TEM (Figure 2b) and DLS (Figure 2d), successfully formed. Attempted coassembly of GroEL10c with GroEL10d also resulted in the formation of NT10c/10d. When the molar ratio of two GroELs was not unity, shorter NTs resulted (Figure S8). Considering also that their polydispersity indexes (PDI) were around 2.0, the coassembly of DNAmodified GroELs, as expected, occurs analogously to a stepgrowth manner. NTs was highly stable thermodynamically and tolerant under chromatographic (SEC) conditions (Figure 2c, black curve). This takes advantage of the fact that the GroEL units in these NTs are strongly bound to one another due to the largely multivalent nature of the coassembly. However, the large multivalency causes a drawback that unfavorable kinetic processes easily compete with the desired thermodynamic process. In fact, when the annealing temperature for the coassembly of GroEL10a and GroEL10b was lowered than 50 °C, only ill-defined agglomerates formed, as visualized by TEM (Figure S9). One may also notice that the GroEL components in NT10a/10b are loosely connected spatially (Figure S10). This is more explicit when compared with the GroEL nanotube obtained using the merocyanine/Mg2+ coordination chemistry, where the GroEL units are densely connected each other.5 Furthermore, the apical domains7 are slightly lifted up probably due to a mechanical shear caused by the DNA hybridization (Figure 2b inset). As shown in Figure 1, DNAs, which are hybridized with the DNA strands at the apical domains of GroEL, can be displaced by externally added DNAs when their sequences are more complementally. We envisioned that this idea could be harnessed for the design of selectively cleavable GroEL nanotubes. For this purpose, we attempted the coassembly of GroEL15c and GroEL10d,6 where the DNA strands in these GroELs are partially complementary to one another, and the connecting parts in NT15c/10d therefore would bear a single-stranded toehold region as depicted in Figure 3a. Thus, 50 mM Tris-HCl buffer solutions (0.3 µM) of GroEL15c and GroEL10d, both containing 100 mM KCl (pH 7.6), were gently heated at 45 °C for 5 min and then mixed together equimolarly. The mixture was slowly cooled to 20 °C at a rate of 0.1 °C/s, whereupon NT15c/10d was successfully obtained, as confirmed by SEC (Figure 3b) and TEM (Figure 3c). Then, NT15c/10d in the resulting solution was incubated with DNA 15d, which is fully complementary to the DNA strand in the GroEL15c component. DLS analysis showed a quick and complete dissociation of NT15c/10d in only 3 min at [15d]/[15c in NT15c/10d] = 5 (Figure S11). The reaction mixture, incubated for 30 min at 37 °C, showed

a bimodal SEC profile (Figure 3b, blue curve) assignable to a mixture of GroEL15c/15d and GroEL10d. This result was supported by a TEM micrograph of the reaction mixture (Figure 3d), where no 1D assembly but only monomeric GroEL was observed. Even at [15d]/[15c in NT15c/10d] = 1, the dissociation of NT15c/10d, as observed by DLS (Figure S11), was completed in only 5 min. As a reference, we likewise incubated NT10a/10b with 10b at [10b]/[10a in NT10a/10b] = 5. However, even after 30 min at 37 °C, no monomeric GroEL but NT10a/10b was observed by SEC and TEM (Figure S12). Namely, by using the DNA hybridization chemistry, NT15c/10d can be cleaved off sequence-specifically. Selective cleavage of NT15c/10d by DNA 15d occurs even in the presence of other GroEL nanotubes such as NT10c/10d. Thus, we mixed fluorescently labeled NT10c/10d carrying Alexa Fluor® 568, non-labeled NT15c/10d (Figure 4a), and 15d in 50 mM Tris-HCl buffer (100 mM KCl, pH 7.6), and incubated the mixture at 37 °C for 30 min. The SEC trace of the reaction mixture, monitored by a UV detector (Figure 4b, black curve), showed two different GroELs. Because none of these GroELs were detectable with a fluorescence detector

Figure 3. (a) Schematic illustration of the formation of a GroEL nanotube (NT) cleavable in a sequence-specific manner, using partially complementary DNA strands for connecting the GroEL monomer units: GroEL15c carrying 15-nt DNA strands of 15c and GroEL10d carrying 10-nt DNA strands of 10d coassemble into nanotube NT15c/10d. This nanotube was mixed with 15-nt DNA 15d, which is fully complementary to the DNA strands of 15c. (b) SEC traces (UV, λ = 280 nm) of GroEL15c (green), GroEL10d (red), and NT15c/10d (an equimolar mixture of GroEL15c and GroEL10d) before (black) and after (blue) the treatment with 15d. (c, d) TEM images of NT15c/10d before (c) and after (d) the treatment with 15d.

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(Figure 4b, green curve), they should originate from NT15c/10d (non-labeled) in consistency with our prediction from Figure 3 that DNA 15d selectively cleaves off NT15c/10d to generate a mixture of GroEL15c/15d and GroEL10d.6 Meanwhile, labeled NT10c/10d remained in the reaction mixture, judging from the luminescence in the higher molecular weight region (Figure 4b, green curve). The coexistence of GroEL nanotubes (NT10c/10d) and monomers (GroEL15c/15d and GroEL10d) in the reaction mixture was confirmed by TEM (Figure 4c). Note that nanotubes that are selectively cleavable by DNAs sequence-specifically are unprecedented. In summary, we have developed a new family of molecular chaperone GroELs, which bear multiple DNA strands on each of their apical domains. These GroELs with complementary DNA strands can coassemble via a largely multivalent interaction, affording protein nanotubes with exceptionally high thermodynamic stabilities. Using partially complementary DNA strands, the nanotubes can be designed to be selectively cleavable while other GroEL nanotubes remain intact. The novel DNA-appended molecular chaperones and their bio-responsive nanotubes, reported herein, have broad potential in pharmacological applications. Their design strategy is expandable for the inclusion of DNA or RNA aptamers that can selectively sense biological cues.8 We envision that our nanotube can be elaborated to serve as a new class of carriers that can function in response to multiple endogenous or transient biological signals. These are the

Figure 4. (a) Schematic illustration of the selective cleavage of NT15c/10d in the presence of NT10c/10d: To an equimolar mixture of fluorescently labeled NT10c/10d and non-labeled NT15c/10d was added 15-nt DNA 15d. (b) SEC traces of the mixture of NT10c/10d and NT15c/10d monitored with UV (λ = 280 nm, black) and FL (λext = 578 nm, λem = 603 nm, green) detectors before (upper charts) and after (lower charts) the incubation with 15d. (c) TEM micrograph of the mixture of NT10c/10d and NT15c/10d after the incubation with 15d.

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interesting subjects worthy of further investigation. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Details of experimental procedures including synthesis of DNA-appended molecular chaperones.

AUTHOR INFORMATION Corresponding Authors

[email protected] [email protected]

ACKNOWLEDGMENTS This work was supported by the Japan Society for the Promotion of Science (JSPS) through its Grant-in-Aid for Specially Promoted Research (25000005) on “Physically Perturbed Assembly for Tailoring High-Performance Soft Materials with Controlled Macroscopic Structural Anisotropy”. S. S. thanks JSPS for Leading Graduate Schools (MERIT) and Young Scientist Fellowship. We thank Dr. Okuro, Mr. Arai, and Mr. Hayashi for their assistance for the experiments.

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