Orthogonal Operation of Constitutional Dynamic Networks Consisting

Nov 15, 2017 - The orthogonal and reversible operations of the CDN DNA tweezers are demonstrated, using T-A·T triplex or K+-stabilized G-quadruplex a...
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Orthogonal Operation of Constitutional Dynamic Networks Consisting of DNA-Tweezer Machines Liang Yue,† Shan Wang,† Alessandro Cecconello,† Jean-Marie Lehn,‡ and Itamar Willner*,† †

Institute of Chemistry, The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel ‡ Institut de Science et d’Ingénierie Supramoléculaires (ISIS), University of Strasbourg, 8 Rue Gaspard Monge, Strasbourg 67000, France S Supporting Information *

ABSTRACT: Overexpression or down-regulation of cellular processes are often controlled by dynamic chemical networks. Bioinspired by nature, we introduce constitutional dynamic networks (CDNs) as systems that emulate the principle of the nature processes. The CDNs comprise dynamically interconvertible equilibrated constituents that respond to external triggers by adapting the composition of the dynamic mixture to the energetic stabilization of the constituents. We introduce a nucleic acid-based CDN that includes four interconvertible and mechanically triggered tweezers, AA′, BB′, AB′ and BA′, existing in closed, closed, open, and open configurations, respectively. By subjecting the CDN to auxiliary triggers, the guided stabilization of one of the network constituents dictates the dynamic reconfiguration of the structures of the tweezers constituents. The orthogonal and reversible operations of the CDN DNA tweezers are demonstrated, using T-A·T triplex or K+-stabilized G-quadruplex as structural motifs that control the stabilities of the constituents. The implications of the study rest on the possible applications of input-guided CDN assemblies for sensing, logic gate operations, and programmed activation of molecular machines. KEYWORDS: nucleic acid, nanobiotechnology, supramolecular structure, DNAzyme, G-quadruplex, triplex, strand displacement

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state Y, where the content of AA′ is enriched by the concomitant separation of constituents AB′ and BA′, and the simultaneous enrichment of the constituent BB′. Treatment of state Y with the counter-trigger T1′ that destabilizes AA′ leads to a system response that recovers state X. Similarly, the T2 triggered stabilization of the constituent AB′ in state X leads to state Z, where constituents AB′ and BA′ are up-regulated, while constituents AA′ and BB′ are down-regulated. As before, destabilization of AB′ by the counter-trigger T2′, recovers state X. CDN ensembles reveal distinct chemical properties reflected by the self-organization and adaptive features and triggered, effector-induced, dynamic programmability. Significant advances in designing chemical CDN assemblies were reported, including the orthogonal triggering of equilibrated hydrazone/ arylhydrazone constituents by light or metal ions11 and the orthogonal control of polymeric hydrazone/hydrazine grids by Lewis acids or metal ions as triggers.12,13 Despite the progress

ntracellular biological transformations are often upregulated or down-regulated by complex dynamically equilibrated mixtures of constitutional networks.1−4 Bioinspired by the natural processes, substantial research efforts are directed to emulate such networks by artificial means.5−8 An artificial constitutional dynamic network (CDN) consists of a mixture of self-assembled molecular or macromolecular constituents that are interconverted by componentexchange. The contents of the constituents in the equilibrated mixture are dictated by the thermodynamic stabilities of the constituents. Subjecting the CDN to an external physical or chemical trigger (effector), which affects the stability of one of the network constituents, leads to a “system response”, whereby the composition, and eventually, the functions of the network are altered to adapt to the input trigger.9,10 The concept of the input control of CDNs is exemplified in Figure 1. The CDN in state X includes four equilibrated constituents, AA′, BB′, AB′, and BA′. While constituents connected with blunt lines share components, the constituents connected with arrows do not share components. Subjecting the mixture to the trigger T1 stabilizes constituent AA′ that results in the system response, © 2017 American Chemical Society

Received: June 29, 2017 Accepted: November 15, 2017 Published: November 15, 2017 12027

DOI: 10.1021/acsnano.7b04557 ACS Nano 2017, 11, 12027−12036

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available.15 These include, for example, fuel/antifuel stimulated strand displacement,16,17 metal-ion bridged bases18,19 (e.g., Ag+ions and ion-eliminating ligands as counter-triggers (e.g., cysteine)), pH,20−22 and light23,24 (e.g., intercalation and removal of trans-azobenzene and cis-azobenzene photoisomers). These triggers provide general means to control adaptive nucleic acid-based CDNs. (iii) A variety of sequencespecific catalytic nucleic acid scaffolds (DNAzymes) are available, such as the hemin/G-quadruplex peroxidase-mimicking DNAzyme25−28 or the metal-ion-dependent DNAzymes.29−31 These catalytic functions of nucleic acids not only introduce functions into the CDNs, but also allow the use of these catalytic reactions of the DNAzymes to provide readout signals for the CDN constituents (e.g., color, fluorescence, and chemiluminescence). These properties of nucleic acids allowed the development of the topic of DNA nanotechnology.32−34 Diverse supramolecular DNA structures acting as switches,14 DNA machines35−37 (e.g., tweezers,38−41 walkers,42−48 or swings49), or reconfigurable 2D50−52 and 3D nanostructures53,54 were produced, and DNA-based materials with switchable macroscopic properties, such as stimuliresponsive hydrogels,55−57 were demonstrated. Different applications of switchable DNA structures were reported,57,58 including their use as sensors, logic gates and computing circuits, stimuli-responsive micro- and nanocarriers for drug release, shape-memory hydrogels, and stimuli-responsive hydrogel actuators or transducers exhibiting programmed mechanical properties. Thus, the rich tool-box of structural and functional information encoded in nucleic acids provides a versatile means to shift the equilibrium mixtures of different nucleic acid-based CDNs and to readout the contents of the constituents by the catalytic and photophysical properties of the constituents. Recently, we highlighted the feasibility to construct nucleic acid-based CDNs,59 and we introduced means to follow the triggered adaptive reconfiguration of the

Figure 1. Trigger-guided reversible switching of constitutional dynamic networks consisting of four interconvertible constituents. T1/T1′ and T2/T2′ represent two orthogonal triggers and countertriggers.

in designing biomimetic CDN systems, the field suffers from several limitations: (i) The diversity of interconvertible chemical constituents is limited and lacks a versatile chemical building block. (ii) The nature of chemical triggers that can be adapted for the operation of the CDNs is limited. (iii) The operation of the CDNs usually lacks a sequential programmable functionality. The base sequence comprising nucleic acids provides a rich “tool-box” to design and construct complex stimuli-responsive supramolecular CDN assemblies: (i) The number and nature of base pairs comprising duplex nucleic acids provide a versatile means to delicately control the stability of equilibrated supramolecular CDN constituents.14 (ii) Many different auxiliary triggers and the respective counter-triggers that stabilize and destabilize duplex nucleic acid structures are

Figure 2. Switchable control of the open/closed tweezer constituents in two CDNs, “K” and “L”, using nucleic acid strand E as effector and E′ as counter-effector. The effector E-induced stabilization of the constituent AB′ in CDN “K” forms the T-A·T-stabilized constituent AB′TAT, leading to CDN “L”. Treatment of CDN “L” with the counter-trigger E′ destabilizes the AB′TAT constituent by the formation of the E/E′ duplex, a process that regenerates CDN “K”. The quantitative evaluation of the contents of the constituents of CDNs “K” and “L” is followed by the catalytic functions of the constituents AA′ and BB′ and the fluorescence features of the tweezers AA′ and AB′ labeled with fluorophore (Cy5, F1)/quencher (IBRQ, Q1) labels and of BA′ and BB′ labeled with fluorophore (Cy3, F2)/quencher (BHQ-2, Q2) labels. The catalytic functions of tweezers AA′ are followed by the hemin/G-quadruplex-catalyzed oxidation of ABTS2− by H2O2 to form ABTS•−, panel i. The catalytic functions of BB′ are followed by the catalytic cleavage of a fluorophore−quencher-modified substrate by the Mg2+-dependent DNAzyme associated with the constituent BB′, panel ii. 12028

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resulting fluorescence provides then, a means to quantify the concentration of BB′. (iii) The fluorophore Cy5 associated with AA′ and AB′ provides a cumulative fluorescence value for the two constituents, while the fluorescence of Cy3, associated with BA′ and BB′ provides a cumulative fluorescence value generated by the two constituents. That is, by extracting the calibration curves of the catalytic activities of intact structures of AA′ and BB′, as a function of their concentrations, the molar concentrations of these constituents in the CDN can be evaluated. Furthermore, by deriving calibration curves relating the fluorescence intensities of the separated intact structures of the CDN (AA′, BB′, AB′, and BA′) as a function of their concentrations, the concentrations of AB′ and BA′ can be evaluated by subtracting the fluorescence intensities contributed by AA′ and BB′, as derived from their concentrations (calculated through the respective DNAzyme-catalyzed transformations) from the cumulative fluorescence values of Cy5 (AA′ + AB′) and Cy3 (BB′ + BA′) and the use of the respective fluorescence calibration curves of AB′ and BA′, respectively. The signal-triggered reconfiguration of the CDN and the transformation of CDN “K” into the CDN “L” are depicted in Figure 2. Subjecting CDN “K” to the effector strand E (9) results in the formation of the T-A·T triplex unit between 9 and the two tethers associated with AB′ to yield the constituent AB′TAT. Formation of the triplex stabilizes this constituent, and the system adapts to an equilibrium, where the concentration of AB′TAT increases and the concentration of the constituent BA′ also increases. Conversely, the effector-induced decrease of the concentrations of AA′ and BB′ is anticipated. Subjecting the CDN “L” to the counter-effector E′ (10) results in the displacement of E (9), the separation of the triplex, and the recovery of CDN “K” from CDN “L”. That is, by the cyclic treatment of the CDNs with the effector 9 and counter-effector 10, the CDN can be switched between CDN “L” and “K” states, respectively, demonstrating the control over the mechanical constituents in the systems and their chemical functionalities. (Note that the transition of CDN “K” to CDN “L” was followed after an equilibration time interval of 12 h, for further discussion vide inf ra.) It should be noted that the effector-induced shifts of the equilibrated transitions of the CDNs between states “K” and “L” yield tweezers constituents exhibiting different “mechanically induced” supramolecular structures. For example, the effector E induced the shift of CDN “K” to “L”, resulting in the increase in the contents of the open tweezers AB′ and BA′ that include “uncaged” single strands L2 and L3. Such single strands may have future potential applications, as they can be designed to act as aptamers that inhibit target proteins. Figure 3a depicts the time-dependent absorbance changes as a result of the hemin/G-quadruplex-catalyzed oxidation of ABTS2− by H2O2 to form ABTS•− before subjecting the CDN “K” to the effector E (i) and after subjecting the CDN “K” to the effector E (transition to CDN “L”) (ii). Similarly, Figure 3b shows the time-dependent fluorescence changes generated by the constituent BB′ before treatment with the effector E (i) and after treatment with the effector E (ii). Evidently, the catalytic activities associated with constituents AA′ (the oxidation of ABTS2− by H2O2 to form ABTS•−) and BB′ (the cleavage of the substrate 11 by the Mg2+-ions-dependent DNAzyme) in CDN “K” are higher than those in CDN “L”, implying that the concentrations of the closed tweezers are higher in CDN “K” as compared to CDN “L”. Figure 3c depicts the cumulative fluorescence changes of Cy3 and Cy5 before the application of

CDNs. In the present study, we further advance the area of DNA nanotechnology by integrating DNA machines into CDNs. We report on the assembly of CDNs consisting of a mixture of DNA tweezers nanostructures. In the presence of appropriate triggers (fuel/antifuel strands or K+ ions), the open/closed states of the tweezers constituents are controlled. We demonstrate the programmed control of the mechanical and chemical functions of the constituents associated with the CDNs. In addition to the control of the “mechanical” properties of the CDNs, we note that the effector(s)-stimulated shifts of the CDNs yield “uncaged” single-stranded tethers coupled to the supramolecular nucleic acid tweezers. These uncaged single-strand tethers add an important functionality into the CDN assemblies, since they could act as functional units, for example, aptamer sequences that inhibit target proteins (vide inf ra).

RESULTS AND DISCUSSION One CDN system composed of four tweezers is depicted in Figure 2. The system is designed to include an equilibrium of two closed tweezers, AA′ (state I) and BB′ (state II), and two open tweezers, AB′ (state III) and BA′ (state IV). While the pairs of tweezers AA′/BB′ and AB′/BA′ do not share components in the respective constituents, the peripheral pairs of tweezers of the CDN constituents AA′/AB′, AA′/BA′, BB′/AB′, and BB′/BA′ share common components. The tweezers AA′ includes as component A the supramolecular structure of strands 1, 2, and 3, where strand 2 bridges the arms 1 and 3. The component A′ is composed of strand 4, and it bridges the arms 1 and 3 by complementary base-pairing that retains the tweezers in the closed state. (Note that the different colors in Figure 2 identify complementary base-pairing domains or defining different functions of the constituents, vide inf ra). The structure in AA′ includes in the arm 3 and strand 4 domains a′ and a (brown) that are guanosine-rich. The strand 1 includes a thymidine-rich tether (yellow), b. Also, the bridging unit 2 is functionalized at its 5′- and 3′-ends with a fluorophore (Cy5) and a quencher unit (IBRQ), respectively. The constituent BB′ is composed of the supramolecular structure comprising the strands 5, 6, and 7, where the arms 5 and 7 are bridged by the fluorophore (Cy3)/quencher (BHQ-2)functionalized strand 6, as component B. The tweezers structure is retained in the closed state II by bridging the arms 5 and 7 with strand 8 (component B′). Also, strand 8 includes the thymidine-rich tether b′ (yellow). The 5′- and 3′ends of strands 7 and 8 include the tethers c and d that correspond to the Mg2+-dependent DNAzyme subunits. The structures AB′ and BA′ include the respective components and are retained in their open configurations, state III and state IV, using appropriate hybridization patterns. Note that the bridges 2 in AB′ (state III) and 6 in BA′ (state IV) are modified with the Cy5/quencher and Cy3/quencher units, respectively. The quantitative distribution of the dynamically equilibrated constituents of the CDN system “K” is probed as follows: (i) the colorimetric assay of the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme, generated by the G-rich subunits a and a′ associated with AA′ in the presence of K+-ions and hemin, which leads to the oxidation of ABTS2− by H2O2 to the colored product, ABTS•− (inset i, Figure 2). (ii) The selfassembly of the tethers c and d, associated with BB′, in the presence of Mg2+-ions yields the Mg2+-dependent DNAzyme that catalyzes the cleavage of its fluorophore-quencher-modified ribonucleobase-containing substrate (Inset ii, Figure 2). The 12029

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fluorescence intensities and derived calibration curves corresponding to the constituents AA′ and BB′. Similarly, Figure S3 shows the calibration curves corresponding to the fluorescence intensities of Cy5 and Cy3 associated with different concentrations of the separate intact constituents AB′ and BA′. Table 1 summarizes the contents (concentrations) of the Table 1. Concentrations of the Constituents Associated with the Dynamic Networks Displayed in Figure 2 CDN state

[AA′], μM

[BB′], μM

[AB′], μM

[BA′], μM

“K” “L”

0.71 ± 0.03 0.28 ± 0.01

0.59 ± 0.02 0.17 ± 0.01

0.16 ± 0.01 0.58 ± 0.01

0.38 ± 0.01 0.90 ± 0.02

different constituents in the CDNs “K” and “L”. Evidently, subjecting CDN “K” to the effector E stabilizes the constituent AB′, resulting in an increase in its content (concentration) in CDN “L” by 263% and the concomitant increase in the concentration of the constituent BA′ by 137%, while the contents of AA′ and BB′ decreased by 61% and 71%, respectively. Figure S4 summarizes the concentrations of the constituents in CDNs “K” and “L” in the form of bars presentation (for further gel electrophoresis experiments examining the different constituents see Figures S5 and S6). The transitions between the CDNs “K” and “L” are reversible, and the treatment of CDN “L” with the counter-effector, E′ (10), regenerates CDN “K”, while the reverse treatment of the CDN “K” with the effector E (9) recovers the CDN “L”. Figure S7 depicts the time-dependent catalytic functions of the constituents AA′ and BB′ and the fluorescence features of the systems upon the switchable transition of E

E′

CDN “K” (i) → CDN “L” (ii) → CDN “K”, (iii). Figure 4 deFigure 3. Probing the dynamic transitions of CDN “K” to CDN “L”: (a) Time-dependent absorbance changes resulting upon the hemin/G-quadruplex-catalyzed oxidation of ABTS2− by H2O2 to form ABTS•− by (i) AA′ constituent in CDN “K” or (ii) AA′ constituent in CDN “L” after treatment of CDN “K” with the effector E. (b) Time-dependent fluorescence changes generated by BB′ upon the cleavage of the substrate 11 by the Mg2+-dependent DNAzyme: (i) BB′ constituent in CDN “K”; (ii) BB′ constituent in CDN “L” generated by subjecting CDN “K” to E. (c) Fluorescence spectra corresponding to the fluorescence of (I) the mixture of constituents BA′ + BB′ in (i) CDN “K” or (ii) CDN “L” after subjecting CDN “K” to the effector E or (II) the mixture AA′ + AB′ (or AB′TAT) in (i) CDN “K” or (ii) CDN “L” after treatment of CDN “K” with effector E.

Figure 4. Concentrations of the constituents associated with the CDNs shown in Figure 2 (in the form of a bar presentation) upon the switchable reconfiguration between states “K” and “L”. State “L” is generated by treatment of the initial state or any “K” state with the effector E, 1 μM. State “K” is generated by subjecting state “L” to the counter-effector E′, 1 μM. The reconfiguration of the CDNs is probed by quantitative evaluation of the content of the constituents by the catalytic and fluorescence features of the constituents and by using appropriate calibration curves, Figures S1−S3.

the effector E, curve i, and after the application of the effector E that transforms CDN “K” into CDN “L”, curve ii. Evidently, low intensities of the two fluorophores in CDN “K” are observed, while the effector E-induced transition of CDN “K” into “L” yields enhanced fluorescence intensities of the two fluorophores, suggesting quantitative enrichment of the constituents AB′ and BA′ (where the spatial separation between the fluorophores and the quenchers is increased in the open configurations of these tweezers constituents). Figure S1a,b shows the calibration curve corresponding to the oxidation of ABTS2− by H2O2 to form ABTS•− by different concentrations of the intact constituent AA′. Figure S1c,d depicts the calibration curve corresponding to the timedependent fluorescence changes generated by different concentrations of the constituent BB′. Figure S2 shows the

picts the switchable effector/counter-effector-stimulated concentrations of the different constituents in the CDNs “K” and “L”. In this experiment, a volume of CDN “K” (450 μL) is prepared and subjected repeatedly to a concentrated solution of the effector E or counter- effector E′ (so that only negligible volume changes of the CDNs occur). In each step of shifting CDN “K” to “L” and back, aliquots of 90 μL are withdrawn 12030

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ACS Nano from the respective CDN mixtures, and these are used to evaluate the contents of the constituents via the respective DNAzyme and fluorescence/quencher reporter units. Evidently, the contents of the constituents in the two CDNs adapt themselves to the information dictated by the effector/ counter-effector: in CDN “K” the constituents AA′ and BB′ predominate, while in the CDN “L” the constituents AB′ and BA′ predominate. (For further support of the transition of CDN “K” to CDN “L” by electrophoretic experiments, see Figures S5 and S6 and accompanying discussion). It should be noted that the time interval for the dynamic equilibration of CDN in state “K” to state “L” corresponded to 12 hours (see Experimental Section for details). This time interval was selected to ensure the complete equilibrium shift between CDNs “K” and “L”. In fact, using shorter time intervals for the equilibration of the transition of CDN “K” to “L”, for example, 4 h, yielded an equilibrium shift of CDN “K” to “L” that corresponded to 85% of the complete equilibration after 12 hours (see Figure S8 and accompanying discussion). It should be noted that the addition of the DNAzyme substrate to the mixture of constituents of CDN “K” has no effect on the contents of the constituents in CDN “K” or on the dynamic equilibration of CDN “K” to “L” (see Figure S9 and accompanying discussion). We note that the addition of K+ions and hemin to the CDNs in states “K” or “L” to quantitatively report the contents of the constituent AA′ via the hemin G-quadruplex-catalyzed oxidation of ABTS2− has no effect on the equilibrium of the CDNs within the time course of the catalytic readout process. Furthermore, the hemin/Gquadruplex-catalyzed reaction proceeds on a time scale of 15 min, whereas the dynamic reconfiguration of the CDN “K” to “L” occurs on a time scale of hours. The lack of effect of the hemin/G-quadruplex on the equilibrium of CDNs “K” and “L” was proved by following the fluorescence of F1 and F2 of CDNs “K” and “L” before and after (15 min) the addtion of K+ ions and hemin. We find that the fluorescence features of F1 and F2 are unchanged, implying that the equilibrated CDNs in states “K” and “L” were not affected by the addition of K+ ions and hemin. It should be noted that the strands comprising the constituents of the CDNs represent optimized sequences for maximum shifts in the triggered reconfiguration of the CDNs (For a detailed description of the design of the sequences and their optimization, see Figure S10, Supporting Information, and the accompanying discussion). Furthermore, the existence of signal-triggered transitions of equilibrated mixtures between states “K” and “L” implies that free components associated with the CDN constituents are present in the respective solutions. Knowing the initial concentrations of the components and the experimental values of the respective components in the constituent structures, we estimated the contents of the free components, and these were used to evaluate the dissociation constant of each of the constituents (For further details see Supporting Information Table S4, and accompanying discussion). We have further examined the orthogonal control of the dynamic equilibrium of the CDNs by the triggered stabilization of the constituents AA′ in CDN “K”, in contrast to the stabilization of the constituent AB′ by the effector, E, in the previous system. (The term “orthogonal control” aims to describe the triggering of the intersecting opposite pairs of constituents in the mixture of four constituents, cf. Figure 1) Toward this end, the CDN “K” was subjected to K+ ions, Figure 5. This results in the formation of the K+-stabilized G-

Figure 5. Control of the constituents of CDNs by the effectorinduced formation of G-quadruplex using K+ ions as effector. Inset i, schematic hemin/G-quadruplex-catalyzed oxidation of ABTS2− by H2O2 to yield the colored ABTS•− (ABTS2− = 2,2′-azinobis(3ethylbenzothiazoline-6-sulfonate)). Inset ii, schematic readout of the catalytic constituent BB′ by the Mg2+-ion-dependent DNAzyme units. CDN “K” and “M” indicate the structures of the constituents in the CDNs before and after subjecting the system to the input K+ ions.

quadruplex between the guanosine tethers a and a′ associated with AA′. The stabilization of AA′ by the formation of the Gquadruplex-stabilized structure, AA′-K+, is anticipated to reconfigure CDN “K” into CDN “M”, where the equilibrated mixture is enriched with AA′-K+ inducing the concomitant enrichment of the constituent BB′, while the concentrations of the constituents AB′ and BA′ decrease. Again, the quantitative evaluation of the concentration of constituents AA′ or AA′-K+ is achieved by the hemin/G-quadruplex-catalyzed oxidation of ABTS2− by H2O2 to form the colored product ABTS•−, while the concentration of BB′ in the two CDNs is probed by the time-dependent fluorescence changes generated by the Mg2+dependent DNAzyme associated with BB′. The concentrations of AB′ and BA′ are evaluated by analyzing the cumulative fluorescence features of Cy5 associated with AA′ + AB′ and of Cy3 associated with BB′ + BA′. By subtracting the calculated fluorescence values contributed by AA′ and BB′, using their catalytically derived concentrations and the respective fluorescence calibration curves of the separate intact AA′ and BB′ constituents, the net fluorescence intensities of AB′ and BA′ were evaluated. By the extraction of calibration curves relating the fluorescence features of the intact constituents AB′ and BA′ as a function of their concentrations, the quantitative evaluation of the concentrations of AB′ and BA′ in the different CDNs was accomplished. Figure S11a,b depicts the calibration curve corresponding to the catalytic rates for generating ABTS•− by the hemin/G-quadruplex DNAzyme associated with different concentrations of the intact AA′ constituent, and Figure S11c,d 12031

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ACS Nano shows the calibration curve corresponding to the catalytic rates of the Mg2+-dependent DNAzyme associated with the constituent BB′ as a function of the concentration of BB′. Figure S12 depicts the calibration curves corresponding to the fluorescence intensities of the intact constituents AA′ and BB′ as a function of their concentrations. Figure S13 depicts the calibration curves corresponding to the fluorescence intensities of the intact constituent AB′ and constituent BA′ as a function of the concentrations of the different constituents. As discussed earlier, the effector-induced shifts of the contents of the supramolecular constituents leads to the enrichment of constituents coupled to functional single-strand units. In the system described in Figure 5, the K+-ion-induced enrichment of the constituent AA′ includes the coupled single-strand L1. This might act as a protein-inhibiting aptamer or a silencing DNA. Figure 6a depicts the time-dependent absorbance changes corresponding to the H2O2-stimulated oxidation of ABTS2− by H2O2 to form the ABTS•− by the hemin/G-quadruplex associated with the constituent AA′ before the interaction with K+ ions (CDN “K”) (i) and after the interaction with K+ ions (CDN “M”) (ii). Similarly, Figure 6b shows the timedependent fluorescence changes generated by the Mg2+dependent DNAzyme associated with constituent BB′ before the interaction with K+ ions (i) and after the interaction with K+ ions (ii). Evidently, the interaction with K+ ions that stimulates the transition of CDN “K” to CDN “M” results in the increase of the closed tweezer constituents AA′ and BB′, reflected by the enhanced catalytic function of the DNAzymes. Figure 6c shows the fluorescence spectra of Cy3 (cumulative fluorescence of BB′ and BA′) and of Cy5 (cumulative fluorescence of AA′ and AB′) before the interaction with K+ ions (curve i) and after the interaction with K+ ions (curve ii), respectively. Using the respective calibration curves corresponding to the catalytic rates for the formation of ABTS•− and the cleavage of the substrate 11 by the Mg2+-dependent DNAzyme, Figure S11, that report the concentrations of AA′K+ and BB′, and using the cumulative fluorescence values of the pairs BB′ + BA′ and AA′ + BB′ and the respective calibration curves, we evaluated the concentrations of the constituents in CDN “K” and CDN “M”. Table 2 summarizes the concentrations of the constituents. Evidently, the treatment of CDN “K” with K+ ions increases the concentrations of AA′ and BB′ by 34% and 22%, respectively, and the concomitant decrease in the concentrations of the open tweezers, AB′ and BA′, by 60% and 29%, respectively, is observed. It should be noted, however, that the K+-triggered transition of CDN “K” to CDN “M” could not be reversed or cycled. High concentration of 18-crown-6-ether is needed to dissociate the K+-stabilized Gquadruplex, and at this level, the equilibrium of all structural constituents is perturbed. For further qualitative evaluation of the concentrations of the constituents of CDN “K” and CDN “M” before and after the interaction with K+ ions by gel electrophoresis, see Figures S5 and S14 and accompanying discussion. An important issue related to the development of the different CDNs rests on the reproducibility to regenerate the equilibrated mixtures. We find that the deviation in the results, in a set of three experiments, is