DNA Rotaxane Hybrid Nanostructures Exhibiting

Letter pubs.acs.org/NanoLett

Au Nanoparticle/DNA Rotaxane Hybrid Nanostructures Exhibiting Switchable Fluorescence Properties Alessandro Cecconello,† Chun-Hua Lu,† Johann Elbaz, and Itamar Willner* Institute of Chemistry and The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *

ABSTRACT: The preparation of a DNA rotaxane consisting of a circular nucleic acid interlocked, through hybridization, on a nucleic acid axle and stoppered by two 10-nmsized Au nanoparticles (NPs) is described. By the tethering of 5-nm- or 15-nm-sized Au NPs on the ring, the supramolecular structure of the rotaxane is confirmed. Using nucleic acids as “fuels” and “anti-fuels”, the cyclic and reversible transition of the rotaxane ring across two states is demonstrated. By the functionalization of the ring with fluorophoremodified nucleic acids in different orientations, the transitions of the rings between the sites are followed by fluorescence quenching or surface-enhanced fluorescence. The experimental results are supported by theoretical modeling.

KEYWORDS: DNA, rotaxane, machine, nanoparticle, fluorescence, switch

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NPs acting as stoppers at the 3′- and 5′-ends of the axle. We demonstrate the controlled, switchable, and reversible translocation of the ring between two states associated with the axle of the dumbbell rotaxane structure, and we highlight that the tethering of a Au NP to the rotaxane ring allows the assembly of predesigned Au NP composites. We further show that the binding of a fluorophore to the rotaxane ring allows the control of the fluorescence features of the systems upon the mechanical translocation of the ring across the two binding states associated with the “axle” of the molecular device. The synthesis of the Au NP-stoppered dumbbell rotaxane is depicted in Figure 1. The nucleic acid (1) is functionalized at its 3′- and 5′-ends with phospho dithiol tethers as anchoring ligands. The DNA ring (2) includes a domain (red) that is complementary to the domain I on the axle (1). The axle (1) was mixed with the ring (2) and the nucleic acid component (3), which is complementary to domain II of the axle and also includes a toehold tether for subsequent strand displacement. The resulting three-component nanostructure was then allowed to hybridize with the nucleic acid (4) that is complementary to domain III of the axle. The resulting four-component nanostructure was further reacted with an excess of 10 nm Au NPs to yield the Au NP dumbbell rotaxane. The system was then purified by gel-electrophoresis. It should be noted that the resulting rotaxane migrates in the gel, upon applying the electrical field at a lower rate as compared to the ring-lacking

nterlocked supramolecular nanostructures, such as catenanes,1 rotaxanes,2 and knots3, have been synthesized and extensively explored in the past two decades. The stimulicontrolled mechanical transitions within these structures were demonstrated,4 and different external stimuli, such as electrical,5 photonic,4,6 or pH7, were used to trigger the molecular machines. Interlocked nucleic acid structures such as catenanes are known to appear in nature,8 and extensive efforts were directed toward the synthesis of interlocked DNA rings such as catenanes9 or rotaxanes.10 Specifically, the synthesis of mechanically programmable and signal-switchable interlocked DNA nanostructures attracted recent research efforts. For example, the reversible strand-displacement-induced transitions of a three-ring catenane across three different configurations were demonstrated.11 Similarly, an interlocked catenane acting as a molecular motor, rotating with controlled directionality across three states by using Hg2+ ions or pH as fuels was demonstrated.12 The use of DNA as a structural element for the construction of nanostructures and, particularly, interlocked nanostructures, is attractive since other nanoelements, such as proteins or metallic nanoparticles, may be positioned on the DNA scaffolds, resulting in new functionalities emerging from their spatial organization. For example, the programmed organization of enzymes on DNA scaffolds allowed the activation of biocatalytic cascades,13 and the distances separating a nucleic acid-bridged Au nanoparticle (NP) dimer were switched by pH-stimulated transitions of the bridge between duplex and i-motif configurations.14 In the present study, we report on the synthesis of a DNA rotaxane nanostructure, where a DNA ring is threaded on a nucleic acid axle and locked in a nonseparable configuration by two Au © 2013 American Chemical Society

Received: October 17, 2013 Revised: November 17, 2013 Published: November 19, 2013 6275

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Figure 1. Stepwise synthesis of a Au NP-stoppered DNA rotaxane nanostructure. Domains I and II are complementary regions to the interlocked ring. Domain II is partially blocked by (3) to prevent the competitive association of the ring to domain II. Domain III is blocked by (4) and prevents the dethreading of the ring prior to the attachment of the Au NPs acting as stoppers for the rotaxane structure. Domain IV of the ring (2) is a sequence-specific domain to hybridize with the (5)-modified 5- or 15-nm-sized Au NPs.

structure (dumbbell) due to the larger size associated with the interlocked ring structure. After purification, the strands (3) and (4) are removed by a strand displacement process (see experimental details and gel electrophoresis results in the Supporting Information). It should be noted that the stepwise hybridization of the nucleic acids (3) and (4) to the axle nucleic acid (1) led to the best yields of the final Au-NP-stoppered rotaxane. In the primary steps, (3) is hybridized with the axle (1), and this presumably lead to partial rigidification of the axle, thus facilitating the interlocking of ring (2) through the singlestranded domain of the axle. We find that hybridization of (3) and (4) with the axle, prior to threading the ring leads to a low yield of the quasi-rotaxane structure. This, presumably, originates from steric barriers and/or electrostatic repulsions of the ring introduced by the duplex domains. Furthermore, we find that the hybridization of strand (4) with the axle prior to the stoppering with the Au NPs is essential. Again, we find that in the absence of (3) and (4) (or one of them), the yield of the Au-NPs-stoppered rotaxane structure is substantially lower. This presumably originates from either the flexible axle structure that allows self-closure on a single NPs or from slower kinetics of binding of the single-stranded axle to the NPs that enables the unthreading of the ring prior to the stoppering process. To confirm the existence of the interlocked rotaxane ring on the 10 nm Au-NP-stoppered rotaxane axle, we used a 15 nm Au NP modified with the nucleic acid (5) as a reporter unit. The nucleic acid (5) is complementary to domain IV of the ring (2) (Figure 2A), and thus, a three-particle, 15-nm-labeled, rotaxane nanostructure is anticipated to be formed. Figure 2A depicts representative images of three-particle nanostructures that are formed. As expected, the 15 nm NP is in between the two 10

Figure 2. (A) Functionalization of the ring associated with the 10 nm Au-NP-stoppered rotaxane nanostructure with a 15 nm Au NP. Representative XHR STEM images of the resulting three NP structures (bar = 20 nm). (B) Functionalization of the ring associated with the 10 nm Au-NP-stoppered rotaxane nanostructure with a 5 nm Au NP. Representative XHR STEM images of the resulting three-NPfunctionalized rotaxane (bar = 20 nm).

nm NPs. By STEM analysis of large areas of the separated nanostructures (e.g., Figure S3), we estimate the yield of the interlocked rotaxane structure to be ≥40%. Note that we derive a yield value of 40%; yet, NP dimers with an interlocked rotaxane ring that did not hybridize with the reporter nucleic acid might exist in the mixture. Hence, the reported yield is a lower limit. Similarly, we have used 5 nm Au NPs modified with the nucleic acid (5), as reporter NPs for the existence of the interlocked rotaxane ring (Figure 2B). Representative images of the three-Au-NP structures are shown in Figure 2B. Clearly, the small NP is positioned in between the two 10 nm Au NPs, acting as stoppers of the rotaxane structure. By STEM imaging of large areas of the resulting Au NPs structures we estimate a yield of ca. 45% of the resulting three Au NPs assemblies, cf. Figure S3, Supporting Information. It should be noted that for the estimation of the yield of the 2 × 10 nm/1 × 5 nm Au NPs structures and of the 2 × 10 nm/1 × 15 nm Au NPs structures we analyzed at least 12 areas (0.15 μm2 each); thus the quoted yield presents a significant statistical analysis. We then examined the reversible, switchable, translocation of the ring across two hybridization sites I and II of the axle, Figure 3A. In this experiment, the rotaxane ring was hybridized with site I, the axle was rigidified with the blocker units B1 and B2 and the site II was hybridized with strand Fu1, to ensure the 6276

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Figure 3. (A) Functionalization of the ring of the Au-NP-stoppered rotaxane nanostructure with the 3′-Cy3-nucleic acid (6). (B) Fluorescence changes of the (6)-functionalized rotaxane upon the dynamic and cyclic shuttling of the ring between sites I and II. At points a, the rotaxane ring positioned at site I is challenged with antifuel 1 (aFu1) and fuel 2 (Fu2). At points b, the system is subjected to antifuel 2 (aFu2) and fuel 1 (Fu1). (C) Functionalization of the ring of the Au-NP-stoppered rotaxane nanostructure with the 5′-Cy3-nucleic acid (7). (D) Fluorescence changes of the (7)-functionalized rotaxane upon the dynamic and cyclic shuttling of the ring between sites I and II. At points a, the rotaxane ring, positioned at site I, is challenged with antifuel 1 (aFu1) and fuel 2 (Fu2). At points b, the system is subjected to antifuel 2 (aFu2) and fuel 1 (Fu1). For the precise definition of the sequences X′ and X″ in the rings see the Supporting Information.

the transition of the ring to site II results in the increased quenching of the fluorophore. By the cyclic translocation of the rotaxane ring from site I to site II, and back, the fluorescence of the reporter unit is switched between lower fluorescence quenching and enhanced fluorescence quenching values, respectively. As the fluorophore in the dumbbell rotaxane is in close proximity to the plasmonic Au NPs, one might anticipate that the fluorescence intensity of the fluorophore might be subjected to plasmonic effects as a result of the close distances separating the fluorophore from the Au NP. As an attempt to identify such plasmonic effects, we designed the system shown in Figure 3C, where the 5′-Cy3-labeled nucleic acid (7) was hybridized with a different domain, X″, on the rotaxane ring, as compared to the previous fluorescence reporter (6). We then translocated the rotaxane ring across sites I and II, using the respective fuel and antifuel strands. Figure 3D depicts the switchable fluorescence properties of the system upon the translocation of the ring from site I to site II, and back. Interestingly, while in the previous system the translocation of the rotaxane ring from site I to II was associated with enhanced quenching of the fluorophore, in the present system the translocation of the ring to site II is accompanied by a fluorescence enhancement. That is, although the transition of the (7)-modified ring from site I to II shortens the distances between the fluorophore and the surface of the NPs, enhanced fluorescence is observed. The phenomenon of surface-enhanced fluorescence was experimentally15 and theoretically addressed.16 The fluorescence intensity of a fluorophore in the vicinity of a plasmonic Au NP is controlled by various parameters, such as the fluorescence quantum yield

location of the ring on site I. Furthermore, the system is subjected to fuel strands, Fu1 and Fu2, and antifuel strands, aFu1 and aFu2, respectively. In the presence of the fuel strand Fu2 and the antifuel strand aFu1, strand displacement of the rotaxane ring from site I, and the concomitant codisplacement of blocker B2 proceed. At the same time, the strand Fu1 is displaced by aFu1 (to form the duplex Fu1aFu1), thus releasing site II. As a result, the rotaxane ring is released from site I, and it is translocated to the vacant position, site II. By subjecting the resulting rotaxane system with the fuel strand Fu1 and the antifuel strand aFu2, the fuel strand Fu2 is displaced (by forming F2aFu2), and the rotaxane ring is displaced from site II. The deblocked site I allows then the assembly of the original rotaxane dumbbell nanostructure, where the rotaxane ring occupies site I. Thus, by the reversible treatment of the system with the respective “fuel” and “anti-fuel” strands, the dictated, cyclic translocation of the rotaxane ring between sites I and II proceeds. To follow the translocation of the ring (2) from site I to II and back, we hybridized the fluorophore (Cy3)-tethered nucleic acid (6) with a complementary sequence X′ that is part of the ring, a sequence that is not participating in the transitions of the ring between sites I and II. The 3′-fluorophore-modified nucleic acid (6) acts as a luminescent reporter for the structural transitions of the ring between sites I and II. Since the distances separating the fluorophore and the Au NPs are different in sites I and II, the effectiveness of fluorescence quenching will be different (vide infra), thus allowing the probing of the mechanical transitions of the rings. Figure 3B depicts the switchable fluorescence changes upon the cyclic translocation of the rotaxane ring from site I to site II, and back. Evidently, 6277

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Figure 4. (A) Calculated fluorophore−Au NP surface distance-dependent fluorescence yields of the Cy3-modified Au NPs rotaxanes. Panel 1: The (6)-functionalized ring. Bars indicate the fluorescence of the system when the rotaxane ring is treated at: orangeposition I; light greenposition II. Panel 2: The (7)-functionalized ring. Bars indicate the fluorescence intensities of the system when the rotaxane ring is located at: orange position I; light greenposition II. (B) Schematic geometrical distances separating the fluorophore Cy3 from the Au NP’s surface of the (6)functionalized rotaxane system positioned at site II (Panel 1) and of the (7)-modified rotaxane nanostructure positioned at site II (Panel 2), respectively.

of the fluorophore, the size of the NPs, the distance separating the fluorophore from the nanoparticle, and the orientation of the dipole of the fluorophore relative to the surface of the NP. Figure 4A shows the calculated fluorescence changes of Cy3 as a function of the distance separating the fluorophore from a 10 nm Au NP (for the details on the parameters for calculating these fluorescence values see Supporting Information). From the calculated curve we conclude that for distances separating Cy3 from the Au NP that are shorter than 5 nm enhanced quenching should occur, whereas in the distance range corresponding to 5−12 nm enhanced fluorescence should proceed (with a maximum value of 20% at 8 nm). Figure 4B shows the geometrically calculated distances separating the Cy3 from the Au NP surface, in the different rotaxane nanostructures. The experimental results are consistent with the calculated values. For the (6)-functionalized ring occupying state II, a separation distance of 2 to 3 nm is estimated, consistent with the enhanced quenching. For the (7)functionalized ring occupying site II we estimate a separation distance of ca. 7 nm, consistent with enhanced fluorescence. For the (6)- or (7)-modified rings occupying site I, the separation distances are estimated to be ca. 12 nm, and negligible effects on the fluorescence yield should be observed. Thus, the different fluorophore-modified rotaxane nanostructures exhibit switchable cyclic transitions between sites I and II, and they reveal switchable fluorescence quenching or fluorescence enhancement upon occupying site II, phenomena that are controlled by the positioning of the fluorophore on the rotaxane ring.

The ability to link a third Au NP to the hybrid dumbbell rotaxane introduces the possibility to tailor nanostructures of enhanced complexities. For example, by using a 15 nm Au NP modified with two thiolated nucleic acids (5), the formation of bis-Au NP rotaxane nanostructures could be envisaged (Figure 5A). Representative STEM images of the 15 nm Au NP crosslinked two dumbbell rotaxanes nanostructures are shown in Figure 5B. We note, however, that the yield of these nanostructures is very low (5%). Nevertheless, with the generation of these nanostructures, and with some added imagination, the future use of such systems as “DNA nanoelevators” or “four-wheel nano-cars” may be envisaged. To conclude, the present study has demonstrated the synthesis of a Au NP dumbbell DNA rotaxane. The rotaxane structure enabled the construction of programmed Au NP assemblies. The switchable translocation of the rotaxane ring across two sites of the axle was demonstrated, and the dynamic control of the fluorescence properties of a fluorophore linked to the rotaxane ring was addressed. Specifically, we demonstrated that, by altering the position of the fluorophore on the rotaxane ring, the switchable fluorescence properties of the fluorophore can be programmed between surface-enhanced fluorescence and surface-quenched fluorescence, upon the translocation of the rotaxane rings on the DNA device. It should be noted that, while numerous rotaxane structures were reported in the area of organic chemistry, the transition of the concept to DNA nanotechnology is new, and the DNA rotaxanes introduce advantages over the synthetic rotaxane structures. Our study demonstrated for the first time the stoppering of rotaxane 6278

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Figure 5. (A) Cross-linking of two 10 nm Au NP stoppered rotaxane systems through the hybridization of a 15 nm Au NP functionalized with two nucleic acids (5), being complementary to the ring. (B) Resulting XHR STEM images of the 10 nm stoppered Au NP rotaxane, cross-linked by the bifunctional (5)-modified 15 nm NP (bar = 50 nm).

no. 267574 under the EC FP7/2007−2013 program. This research is performed under the auspices of the Minerva Center for Biohybrid Complex Systems.

structures by Au NPs, the programmed switchable and reversible transition of the ring on distinct sites of the axle, the ordered formation of Au NPs structures and the plasmonic control of fluorescence functions by the mechanical operations of the DNA device.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and purification of the ring, synthesis of the Au-NPstoppered rotaxane, electrophoresis experiments, and STEM images. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 972-2-6585272. Fax: 9722-6527715. Author Contributions †

A.C. and C.-H.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported in part by the Israel Science Foundation and by the NanoSensoMach ERC Advanced Grant 6279

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