Letter pubs.acs.org/NanoLett
Switchable Catalytic DNA Catenanes Lianzhe Hu,† Chun-Hua Lu,† and Itamar Willner* Institute of Chemistry, Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *
ABSTRACT: Two-ring interlocked DNA catenanes are synthesized and characterized. The supramolecular catenanes show switchable cyclic catalytic properties. In one system, the catenane structure is switched between a hemin/G-quadruplex catalytic structure and a catalytically inactive state. In the second catenane structure the catenane is switched between a catalytically active Mg2+-dependent DNAzyme-containing catenane and an inactive catenane state. In the third system, the interlocked catenane structure is switched between two distinct catalytic structures that include the Mg2+- and the Zn2+-dependent DNAzymes. KEYWORDS: DNAzyme, hemin/G-quadruplex, chemiluminescence, machine, DNA switch
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reconfigurable bicatalytic catenane system that is switched ON/ OFF between alternate DNAzyme units. The general approach involves the use of two-ring interlocked catenanes composed of a large ring α and a smaller ring β (for the synthesis and purification of the catenane systems see Figure S1 and Figure S2 to S4, Supporting Information). The transition of ring α along ring β yields the catalytic catenane state. The first switchable catalytic system is presented in Figure 1A. Ring α1 includes the sequence I (blue) that corresponds to the G-quadruplex sequence and the domain II consisting of two subsequences (x) (part of the G-quadruplex sequence) and (y). The sequence II is complementary to the domain II′ of ring β1. The domain III of ring α1 includes two subsequences (z) and (w). The domain (w) of ring α1 is complementary to the domain II′ of ring β1 and the extend bases (p) and (q) of ring β1 (that is w is complementary to the sequence q + II′ + p). The strand L1, is partially complementary to the sequence (z) and part of (w) of ring α1, and it acts as a blocker unit. It should be noted that hybridization of the domain (q + II′ + p) of ring β1 with domain (w) of ring α1 is energetically favored as compared to the hybridization of the domain II/II′ of rings α1/β1. Nonetheless, in the presence of the blocker L1 that hybridizes with domains (z) and part of (w), hybridization of ring β1 with the domain (w) of α1 is prohibited. This results in state A, where ring β1 hybridizes with domain II to yield the energetically less-favored duplex II/ II′. Note that the hybridization of ring β1 with domain II blocks part of the G-quadruplex sequence, thus eliminating the formation of the G-quadruplex and the resulting hemin/Gquadruplex DNAzyme. Treatment of state A with the antiblocker strand L1′ results in the displacement of the blocker while forming the stable L1/L1′ duplex. The release of the blocker L1 turns the sequence (w) free, resulting in the
he reconfiguration of DNA nanostructures provides the basis to construct stimuli-responsive DNA switching devices.1 Different external triggers were used to reversibly stimulate DNA structures between distinct two states. These included the K+/crown ether-induced transitions between Gquadruplex and random coil strands,2 the metal ion/ligand transition between metal-ion-bridged and single strand structures,3 the pH-induced transitions between i-motif single strands and duplex structures,4 and the use of temperature.5 Also, DNA machines were switched between two states using, pH as trigger,6 photoisomerizable azobenzene intercalator units,7 and ion/ligand-stimulated mechanical closure and opening of DNA tweezers.6 Interlocked DNA rings−catenanes attracted recent interest as switchable supramolecular devices. Using pH, metal ions/ligands, and nucleic acid fuel/antifuel strands, different switchable DNA catenated devices were demonstrated.8 For example, DNA rotors driven across three distinct states9 and a pH-driven pendulum cycled between two states10 were reported using DNA catenane nanostructures. The design of switchable functions originating from the dynamic reconfiguration of DNA catenanes are, however, scarce. Recently, the switchable cyclic control of fluorescence quenching and surface-enhanced fluorescence of fluorophore/ Au nanoparticle pairs, associated with catenane nanostructures, was demonstrated upon the programmed dynamic reconfiguration of the catenane structures.11 Similarly, the reversible reconfiguration of Au NPs structures using DNA catenanes as mechanical scaffolds for assembling the nanoparticles was demonstrated.12 In the present article we describe the synthesis and characterization of switchable catalytic catenanes. We describe two systems that lead to the ON/OFF switchable catalytic functions, and one system that is switched between two different catalysts, using two-ring catenanes as mechanical reconfigurable devices. As far as we are aware, these are the first examples demonstrating catalytic functions of catenated DNA nanostructure. Particularly interesting is the demonstration of a © XXXX American Chemical Society
Received: December 30, 2014 Revised: January 28, 2015
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DOI: 10.1021/nl504997q Nano Lett. XXXX, XXX, XXX−XXX
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ring β1 from site (w), resulting in the retransition of ring β1 to the energetically less-favored site II of ring α1. This process dissociates the G-quadruplex, leading to the catalytically inactive catenane structure, state A. By the cyclic treatment of the system with L1 and L1′, the catenane structure is switched between catalytically inactive and active states (states A and B, respectively). Figure 1B depicts the chemiluminescence spectra of the switchable catalytic catenane system. In state A, a very low chemiluminescence spectrum is observed, curve (a), that is very close to the background chemiluminescence generated by hemin in the presence of luminol/H2O2. Treatment of state A with L 1 ′ results in state B, and this lead to the chemiluminescence spectrum shown in curve (b). Subjecting state B to strand L1 and the subsequent treatment of the resulting state A with L1′ leads to the switchable deactivation and activation of the hemin/G-quadruplex catalytic catenane, Figure 1B, curves (c) and (d), respectively. The switchable and cyclic chemiluminescence properties of the catalytic catenane are depicted in Figure 1B, inset. The switchable catalytic functions of the catenane were also followed by the hemin/Gquadruplex-catalyzed oxidation of ABTS2− by H2O2 to form the colored product ABTS•−, Figure 1C. Here the experiment is initiated in state B, where the hemin/G-quadruplex catalyzes the oxidation of ABTS2− by H2O2. At point (a), L1 is added to the system. This leads to the formation of state A and to the blockage of the catalytic oxidation of ABTS2−. At point (b), the strand L1′ is added to the system, resulting in the regeneration of the catalytic hemin/G-quadruplex catenane that catalyzes the oxidation of ABTS2− by H2O2. By the repeated treatment of the system with L1 and L1′ the system is switched between state A (OFF) and state B (ON). It is should be noted that the rate of ABTS•− formation in each cycle decreases due to the consumption of the ABTS2−/H2O2 substrates. The second switchable catalytic DNA catenane makes use of the Mg2+-dependent DNAzyme,14 Figure 2A. The catenane system consists of the two rings α2 and β2. The ring α2 includes the domain I that corresponds to the Mg2+-dependent DNAzyme sequence. Domain II of ring α2 includes part of the DNAzyme sequence (i) and additional domain (j). Domain II′ of ring β2 is complementary to domain II. The ring α2 includes also the site III that includes the sequences (k) and (t). The sequence (t) is complementary to the domains (m + II′ + n) of ring β2, implying that hybridization of ring β2 is energetically favored upon binding to (t) as compared to domain II of ring α2. The hybridization of L2 with the sequence (k) and part of (t) prohibits the hybridization of ring β2 with site (t), resulting in the catalytically inactive structure, state A. Treatment of state A with the strand L2′ displaces the strand L2 in the form of the stable duplex L2/L2′, and this results in the reconfiguration of ring β2 to domain (t) that yields the energetically stabilized duplex (t)/(m + II′ + n). The release of ring β2 from site II yields the free sequence I, and this, in the presence of the fluorophore/quencher functionalized substrate S1, self-assembles into the Mg2+-dependent DNAzyme/S1 catenane system. This results in the catalytic cleavage of the substrate, and the fluorophore-functionalized fragment product provides the readout signal for the catalytic catenane, state B. Subjecting state B to strand L2 displaces ring β2 from site (t), while generating the energetically stabilized duplex L2/(k) + part (t). The released β2 ring reconfigures to the energetically less stable position II, a process that separates the Mg2+dependent DNAzyme and its substrate and yields the catalytically inactive catenane state A. By the cyclic treatment
Figure 1. (A) Schematic switchable and cyclic reconfiguration of a DNA catenane system between a catalytically inactive catenane structure, state A, and a hemin/G-quadruplex, catalytically active catenane, state B, using L1/L1′ as blocker/displacer strands. (B) Chemiluminescence spectra generated by the system, in the presence of hemin and luminol/H2O2. (a) In state A. (b) After treatment of state A with L1′. (c) After treatment of the resulting state B with L1. (d) After subjecting the reconfigured state A with L1′. Inset: Cyclic chemiluminescence intensities (at λ = 415 nm) generated upon the reconfiguration of the system between states A and B. (C) Timedependent absorbance changes, originating from the hemin/Gquadruplex-catalyzed oxidation of ABTS2− by H2O2 to form ABTS•− (λ = 420 nm) upon the L1/L1′ stimulated cyclic reconfiguration of the catenane system between states A and B. The measurement is initiated with the catalytically active state B, resulting in an increase in the absorbance. At point (a), the strand L1 is added, resulting in the catalytically inactive structure state A. At point (b), strand L1′ is added to the system, leading to the reconfiguration of state A to state B, and the formation of the catalytically active structure, state B. By the cyclic treatment of the system with L1′ and L1, the system is switched between ON and OFF states, respectively.
reconfiguration of ring β1 to site (w), where a duplex of enhanced stability (w)/(p + II′ + q) is formed. The release of ring β1 from domain II of ring α1 results in the free domain I that self-assembles into the G-quadruplex. The association of hemin to the G-quadruplex yields the hemin/G-quadruplex DNAzyme, state B, that catalyzes the oxidation of luminol by H2O2 to yield chemiluminescence (λ = 415 nm)13 or the oxidation of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid, ABTS2−, by H2O2 to the colored product ABTS•− (λ = 420 nm). Treatment of state B with the blocker unit L1 displaces B
DOI: 10.1021/nl504997q Nano Lett. XXXX, XXX, XXX−XXX
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Figure 2. (A) Cyclic switchable reconfiguration of a two-ring catenane nanostructure between a catalytically inactive configuration, state A, and a catalytically active configuration, state B, that contains the Mg2+dependent DNAzyme. The transition between the states are induced by the L2′ and L2 strands. The reconfiguration of the catenane system is probed by the Mg2+-dependent DNAzyme cleavage of the substrate S1 and the generation of fluorescence. (B) Time-dependent fluorescence changes upon the cyclic reconfiguration of the catenane by the strand L2 and L2′. The measurement is initiated with the system in state B, resulting in the time-dependent fluorescence increase as a result of the DNAzyme-simulated cleavage of S1. At time marked (a), strand L2 is added to the system, resulting in the reconfiguration of the system to state A and the blockage of the catalytic functions of the system. At time marked (b), L2′ is added to the system, leading to the regeneration of state B and the recovery of the DNAzyme that catalyzes the cleavage of S1. By the cyclic treatment of the system with L2 (a) and L2′ (b), the “OFF” and “ON” catalytic states of the catenane are formed.
Figure 3. (A) Switchable, cyclic, reconfiguration of a two-ring catenane system between the Mg2+-dependent DNAzyme, state A, and the Zn2+-dependent DNAzyme, state B, using L3 and L3′ strands as reconfiguration promoters. The Mg2+-dependent DNAzyme cleaves substrate S1 leading to the fluorescence of F1, while the Zn2+dependent DNAzyme cleaves substrate S2, leading to fluorescence of F2. (B) Time-dependent fluorescence changes of F1 (red curve 1) and of F2 (blue curve 2) upon the treatment of the system with L3 and L3′. The measurement is initiated in state A, leading to the increase in the fluorescence of F1 and to an unchanged fluorescence F2. At the time marked (a) L3 is added to the system, resulting in the reconfiguration of state A to state B, a process that switches off the Mg2+-dependent DNAzyme accompanied by the increase of the fluorescence of F2. At time (b), L3′ is added to the system, resulting in the reactivation of state A and the fluorescence increase of F1. By the cyclic treatment of the system with L3 and L3′, the fluorescence is switched between F2− ON/F1−OFF and F1−ON/F2−OFF, respectively.
of state A with L2′ and subsequent states with L2, the catenane system is cycled between the catalytically active catenane, state B, and the catalytically inactive nanostructure, state A. The catalytic ON/OFF functions of the catenane are then followed by the fluorescence of the fragmented substrate formed upon the hydrolytic cleavage of S1. Figure 2B depicts the timedependent fluorescence changes of the system upon the cyclic activation/deactivation of the catalytic functions of the catenane. The measurement is initiated in state B of the catenane assembly. The time-dependent increase in the fluorescence corresponds to the cleavage of S1 by the catalytically active catenane. At point (a) L2 is added to the system, resulting in the reconfiguration of the system to state A, the catalytically inactive catenane. At point (b) the strand L2′ is added to the system, and this regenerates the catalytically active catenane. Finally, the two-ring catenane system was switched between two catalytically active DNAzymes, the Mg2+-dependent DNAzyme and the Zn2+-dependent DNAzyme,15 using the two-ring catenane as scaffold, Figure 3A. The catenane system is composed of rings α3 and β3. In state A, the Mg2+-dependent DNAzyme sequence I exists in a free structure, capable to bind the substrate S1, and ring β3 is hybridized with domain III of ring α3. The domain III is composed of the sequence (d), part of the Zn2+-dependent DNAzyme, and the sequence (e). In the
presence of the strand L3, that reveal complementarity to domains (e + f) of ring α3, the displacement of ring β3 proceeds, resulting in the hybridization of ring β3 with the domain (g + h) associated with ring α3. Note that the sequence (d + e) includes a higher number of bases as compared to (g + h). Thus, the hybrid between the domain III′/(d + e) is energetically stabilized over the hybrid III′/(g + h). That is, in the absence of L3, state A is energetically favored, but upon the L3 stimulated displacement of ring β3, it hybridizes with the energetically less favored site (g + h). This results in the capping of part of the Mg2+-dependent DNAzyme sequences, the inactivation of the DNAzyme, and the assembly of the free Zn2+-dependent DNAzyme sequence II on ring α3, which binds S2 and leads to the formation of state B. The substrate S1 is functionalized with the fluorophore (F1 = ROX) and the quencher (Q1 = BHQ2) units, and the substrate S2 is functionalized with the fluorophore (F2 = FAM) and the quencher (Q2 = Iowa black FQ) units. The close proximity between the fluorophore/quencher pair leads to the quenching of the fluorophore F1 and F2 in the substrate structures. The Mg2+-dependent DNAzyme cleavage of S1 leads to the F1C
DOI: 10.1021/nl504997q Nano Lett. XXXX, XXX, XXX−XXX
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