Transient Dynamic Operation of G-Quadruplex-Gated Glucose Oxidase-Loaded ZIF-90 Metal–Organic Framework Nanoparticle Bioreactors

Glucose oxidase-loaded ZIF-90 metal–organic framework nanoparticles conjugated to hemin-G-quadruplexes act as functional bioreactor hybrids operating transient dissipative biocatalytic cascaded transformations consisting of the glucose-driven H2O2-mediated oxidation of Amplex-Red to resorufin or the glucose-driven generation of chemiluminescence by the H2O2-mediated oxidation of luminol. One system involves the fueled activation of a reaction module leading to the temporal formation and depletion of the bioreactor conjugate operating the nickase-guided transient biocatalytic cascades. The second system demonstrates the fueled activation of a reaction module yielding a bioreactor conjugate operating the exonuclease III-dictated transient operation of the two biocatalytic cascades. The temporal operations of the bioreactor circuits are accompanied by kinetic models and computational simulations enabling us to predict the dynamic behavior of the systems subjected to different auxiliary conditions.

−3 Particularly the information encoded in the base sequence of nucleic acids provides a versatile means to dynamically reconfigure the biopolymer by a rich "tool-box" of triggers into diverse structural motives. 4−33 Different triggers controlling the temporal transient operation of the circuits were introduced, including the application of enzymes, such as endonuclease, 34 nickase, 35 or ligase, 36,37 as catalytic units regulating the formation and temporal depletion of DNA structures, the application of DNAzymes, 38 the ion-induced formation and dissociation of G-quadruplexes, 39 the ribonuclease (RNase)-stimulated control over transcription machineries, 40 or the application of light-signals as temporal regulating stimuli of DNA circuits. 38,41dentification of useful applications of transient dissipative DNA circuits is, however, still challenging.Several applications of transient nucleic acid-based circuits were demonstrated, including the temporal aggregation/deaggregation of metal nanoparticles or semiconductor quantum dots and control over their optical properties of the systems, 42,43 the temporal formation and separation of DNA nanostructures, such as microtubules or wires, 44 and the conjugation of the DNA circuits to enzymes, driving transient biocatalytic cascades. 45evelopment of catalytic nanoparticles, 46−48 such as metal, 49−52 metal oxide, 53−58 metal−organic framework nanoparticles, 59−63 or carbon-based nanoparticles, 64−66 "nanozymes", attracts substantial research efforts in catalysis and materials science.Also, integration of biocatalysts and catalytic nanoparticles into nanoparticle frameworks generated hybrid composites acting as bioreactor systems driving catalytic cascades. 67Tethering of aptamer strands to nanozymes led to aptananozymes revealing the binding and catalytic functions mimicking native enzymes 68 and to cell-targeting catalytic nanoparticles for therapeutic applications. 69Moreover, catalytic nanoparticles conjugated to catalytic nucleic acids, DNAzymes, acted as hybrids operating as bioreactor systems driving catalytic or biocatalytic cascades. 70−78 Particularly, the loading of metal−organic framework nanoparticles with nanoparticle catalysts, 79 biocatalyst, 80 or drugs 81 and capping the loaded nanocarriers with stimuli-responsive nucleic acid gates 82 for controlled release of the loads were demonstrated.
Accordingly, the conjugation of transiently operating nucleic acid gates to nanozymes could introduce dynamic dimensions to nucleic acid/nanozyme hybrids.Here, we report on the assembly of supramolecular bioreactor systems consisting of nucleic acid-modified glucose oxidase (GOx)-loaded ZIF-90 metal−organic framework nanoparticles (NMOFs) and hemin-G-quadruplex constituents demonstrating transient-peroxidase like activities in the presence of the nickase, Nt.BbvCI, or exonuclease III, Exo III, as control units.The ZIF-90 nanoparticles were selected due to the ease to integrate GOx into the ZIF framework and due to the surface carboxaldehyde functionalities of ZIF-90 that allow the covalent conjugation of the nucleic acids to the scaffold. 70TRANSIENT NICKASE-DRIVEN BIOCATALYTIC    As an attempt to understand the effect of these auxiliary parameters on the transient process, we formulated a kinetic model that follows the transient process, Figure S8.This model was adopted to simulate the experimental results (dotted transient points a) in Figure 3B, panel I, with a computationally fitted transient (solid transient curve a′), generated by the set of rate constants comprising the model, that are summarized in Table S1 (for a detailed description of the simulation process see Figure S8).This set of rate-constants has a value provided that it can predict the transient behavior of the supramolecular bioreactor at other auxiliary conditions.Accordingly, the transient behavior of the formation and depletion of (1)+( 2

BIOCATALYTIC CASCADES USING GOX-LOADED/HEMIN-G-QUADRUPLEX-CONJU-GATED ZIF-90 AS FUNCTIONAL FRAMEWORKS
An alternative transient GOx-loaded ZIF-90 NMOFs/hemin-G-quadruplex reaction module leading to the fuel-triggered and transient operation of the bioreactor catalyzing the biocatalytic oxidation of Amplex-Red to resorufin or the biocatalytic generation of chemiluminescence is depicted in Figure 4.In this system, exonuclease III, Exo III, acts as the enzyme controlling the dissipative, transient operation of the bioreactor.The reaction module consists of the GOx-loaded ZIF-90 NMOFs functionalized with the nucleic acid A 2 , (6) (loading of GOx 82 μg/mg NMOFs; loading of A 2 10 nmol/ mg NMOFs), and the hemin-G-quadruplex B 2 , (7).The enzyme Exo III is also included in the reaction module.Subjecting the reaction module to the fuel strand T 2 , (8), results in the assembly of the ( 6)+( 7)/(8) supramolecular bioreactor complex consisting of the ( 8)-bridged GOx-loaded ZIF-90 NMOFs and hemin-G-quadruplex constituents.The supramolecular complex operates two different biocatalytic cascades.One biocatalytic cascade, panel I, involves the aerobic GOx-catalyzed oxidation of glucose to yield gluconic acid and H 2 O 2 and the subsequent hemin-G-quadruplexcatalyzed oxidation of Amplex-Red to resorufin.The second biocatalytic cascade driven by the ( 6)+( 7)/(8) supramolecular GOx-loaded ZIF-90 NMOFs/hemin-G-quadruplex bioreactor involves the aerobic GOx-loaded ZIF-90 NMOFs-catalyzed oxidation of glucose to gluconic acid and H 2 O 2 and the subsequent hemin-G-quadruplex catalyzed oxidation of luminol by H 2 O 2 and the generation of chemiluminescence, panel II.The Exo III included in the system selectively degrades the 3′-ended fuel strand T 2 , leading to the separation of the supramolecular complex regenerating the rest of the reaction module and producing base fragments T 2 as waste products.The separation of the supramolecular complex by Exo III leads to the temporal activation of the biocatalytic cascades shown in panels I and II and to the recovery of the parent reaction module.That is, the T 2 -triggered activation of the reaction module leads to the temporal and transient formation of the ( 6)+( 7)/(8) supramolecular complex consisting of the GOx-loaded ZIF-90 NMOFs/hemin-Gquadruplex that guides the temporal biocatalytic cascades shown in Figure 4, panels I and II.
It should be noted that in order to operate the Exo III driven transient generation of the bioreactor, the supramolecular GOx-loaded ZIF-90 NMOFs/hemin-G-quadruplex complex had to be engineered to retain an intact, Exo III-resistant structure, to the extent that only the T 2 that forms the duplex with B 2 is being digested by Exo III.We find that it is essential to tether to the G-quadruplex B 2 a single strand 3′-terminated tether; otherwise, the G-quadruplex is also degraded by Exo III.
Panels I and II of Figure 5A depict the time-dependent fluorescence changes of resorufin generated upon subjecting the reaction module shown in Figure 4 to the fuel strand T 2 and withdrawing at time interval samples driving the glucoseinitiated H 2 O 2 -mediated oxidation of Amplex Red to resorufin.The withdrawn samples reveal a temporal initial increase in the oxidation rates of Amplex-Red for a time interval of ∼2 h, and afterward, the withdrawn sample shows a continuous temporal decline in the bioreactor catalyzed oxidation rates, which are fully blocked after 8 h, reaching the parent background rates of Amplex-Red oxidation to resorufin by the parent reaction module.Readdition of the fuel strand T 2 to the system reactivated the transient operation of the reaction module, following the oxidation of Amplex-Red to form resorufin.These results are consistent with the T 2 -fueled/Exo III-guided operation of the reaction module shown in Figure 4.The bioreactor Exo III-driven, T 2 -triggered, transient formation of ( 6)+( 7)/(8) supramolecular bioreactor generating fluorescent resorufin is controlled by the concentrations of T 2 , Figure 5B, panel I, and Exo III, Figure 5B, panel II.As the concentrations of T 2 increases, the peak rates of temporal resorufin formation are higher, and as the concentration of Exo III increases, the peak rates of catalyzed formation of resorufin are lower and the depletion of the catalytic rates generating resorufin is enhanced.Figure S11 formulates the kinetic model corresponding to the transient operation of the T 2 /Exo III driven (6)+( 7)/(8) supramolecular GOx-loaded NMOFs/hemin-Gquadruplex bioreactor system.Figure 5B, panel I, curve a′, depicts the fitted, computationally simulated, temporal concentrations of the catalytic ( 6)+( 7)/(8) supramolecular structure generating resorufin by the T 2 /Exo III-driven bioreactor system, overlapping the experiment transient curve a, using the kinetic models.The derived rate constants following the kinetic model are summarized in Table S2.Using this set of rate constants, the predicted curves corresponding to temporal transient concentrations of the catalytic bioreactor system at different concentrations of T 2 and Exo III were computed, curves b′ and c′, Figure 5B, panel I, and d′ and e′, Figure 5B, panel II, and the computational results are experimentally validated, curves b and c, Figure 5B, panel I, and curves d and e, Figure 5B, panel II.Indeed, the experimental results fit well to the predicted transient systems.Moreover, the T 2 /Exo III-driven activation of the GOx-loaded ZIF-90 NMOFs/hemin-G-quadruplex bioreactor, Figure 4, panel II, was applied to stimulate the temporally catalyzed generation of chemiluminescence.Panels I and II of Figure 5C depict the temporal chemiluminescence spectra generated by samples withdrawn at time intervals from the T 2 /Exo IIIactivated reaction module.The triggered reaction module reveals intensified chemiluminescence spectra for a time interval of 2 h and, subsequently, temporal depletion and blockage of the chemiluminescence after 8 h. Figure 5C, panel III shows the temporal, transient concentrations of ( 6)+( 7)/ (8) supramolecular structure generating chemiluminescence upon the T 2 /Exo III-triggered activation of the bioreactor system in the presence of 3 μM T 2 and 0.4 μM Exo III. Figure 5D depicts the experimental and simulated temporal, transient formation of the ( 6)+( 7)/(8) supramolecular bioreactor leading to chemiluminescence upon activation of the reaction module at different concentrations of T 2 and Exo III.As the concentration of T 2 increases, the temporal peak chemiluminescence intensity is higher, and as the concentration of Exo III is elevated, the peak chemiluminescence intensity is lower and the dissipative depletion of the bioreactor driven process is enhanced.

■ CONCLUSION
The study introduced nucleic acid-modified/GOx-loaded ZIF-90 conjugates as functional assemblies guiding transient biocatalytic cascades.Such frameworks could find important nanomedical applications for dose-controlled, temporal release of therapeutic agents.For example, the GOx-stimulated Nano Letters activation of the hemin-G-quadruplex peroxidase-mimicking DNAzyme yields reactive oxygen species (ROS) that might act for temporal chemodynamic treatment of cancer cells. 69lternatively, the triggered temporal unlocking of the NMOFs could be used for the dose-controlled release of drugs, e.g., insulin from the NMOFs carriers. 85Moreover, the present study introduced the fueled strand displacement principle and coupled enzyme driven transformation as control motives of the dynamic process.Other triggering stimuli for temporal operation of the reaction modules such as miRNA/ RNase, 86 redox-triggered aptamer-ligand complexes, 87 or light, 38 may be envisaged.

■ ASSOCIATED CONTENT
* sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.nanolett.3c02542.Experimental section; calibration curves for determination of the loading of GOx and DNA strands A 1 or A 2 involved in the bioreactor; efficient biocatalytic cascades performed by (1)+(2)/(3) supramolecular structure; effect of loading amount of GOx on the transient biocatalytic cascades; calibration curve for determination of the concentration of (1)+(2)/(3) supramolecular structure; kinetic model and simulation details of Nt.BbvCI-guided dissipative bioreactor system; calibration curve for determination of the concentration of ( 6)+( 7)/(8) supramolecular structure; kinetic model and simulation details of Exo III-guided dissipative bioreactor system (PDF)
)/(3) supramolecular bioreactor generating fluorescent resorufin was predicted at different auxiliary concentrations of L 1 ′ (curves b′ and c′, Figure 3B, panel I) and the nicking enzyme concentrations (curves d′ and e′, Figure 3B, panel II).The predicted results were experimentally validated, curves b and c in Figure 3B, panel I and curves d and e in Figure 3B, panel II, respectively.Very good fit between the experimental results and the computationally predicted transients is demonstrated, supporting the kinetic model.Similarly, Figure 3C, panels I and II, depicts the temporal chemiluminescence spectra generated by samples withdrawn at time intervals from the transient operating bioreactor system according to Figure 2, panel II.The L 1 ′-triggered activation of the reaction module yields in the first 2 h of operating the bioreactor system reaction samples where the resulting chemiluminescence is temporally intensified, and afterward the bioreactor samples withdrawn from the reaction system show a continuous temporal decrease in the chemiluminescence, reaching the parent chemiluminescence intensities after a time interval of ∼10 h.These results are consistent with the temporal L 1 ′-triggered buildup of the (1)+(2)/(3) supramolecular GOx-loaded NMOFs/hemin-G-quadruplex catalytic bioreactor system that undergoes transient depletion by the nickase-induced separation of the bioreactor conjugate, leading to the recovery of the original reaction module.The transient concentration changes of (1)+(2)/(3) supramolecular bioreactor generating chemiluminescence are displayed in Figure 3C, panel III.As before, decreasing the concentration of the fuel strand L 1 ′ that activates the reaction module results in lower temporally generated yields of the bioreactor conjugate, leading to lower chemiluminescence intensities, Figure 3D, panel I. Also, increasing the concentration of the nicking enzyme results in lower yields of chemiluminescence generated by the bioreactor system and enhanced depletion rates of the transiently operated bioreactor intermediate, Figure 3D, panel II.

Figure 5 .
Figure 5. (A) Time-dependent fluorescence changes of resorufin generated by samples withdrawn, at time intervals, from the dynamic reaction module depicted in panel I of Figure 4: panel I, samples withdrawn at (a) t = 0 h, (b) t = 1 h, (c) t = 2 h; panel II, samples withdrawn at (d) t = 4 h, (e) t = 6 h, (f) t = 8 h, (g) t = 10 h; panel III, temporal, transient concentration changes of the (6)+(7)/(8) supramolecular structure generating the fluorescent resorufin, upon operation of the dynamic reaction module shown in Figure 4, panel I.The experimental conditions operating the dynamic process shown in panels I−III are A 2 -(6), 1 μM; B 2 -(7), 1 μM; T 2 -(8), 3 μM; and Exo III, 0.4 μM.(B) Probing the effects of the concentrations of the fuel strand T 2 (panel I) and of the Exo III (panel II) on the temporal transient generation of the (6)+(7)/(8) supramolecular structure, generating fluorescent resorufin according to Figure 4, panel I. Part B, panel I: (a) Dotted points correspond to experimental data recorded at the conditions specified in (A); (a′) solid curves correspond to computationally simulated results using the kinetic model formulated in Figure S11, Supporting Information.(b′, c′) Computationally simulated transient concentrations of (6)+(7)/(8) supramolecular structure, generating fluorescent resorufin, at auxiliary conditions T 2 = 2 and 4 μM, respectively; (b) and (c) Dotted data correspond to experimentally validated results in the presence of T 2 = 2 and 4 μM, respectively.Part B, panel II: (a/a′) is the same condition with (a/a′) in panel I. (d′, e′) Computationally simulated transient concentrations of (6)+(7)/(8) supramolecular structure, generating fluorescent resorufin, at auxiliary conditions Exo III = 0.6 and 0.2 μM, respectively.(d, e) Dotted data correspond to experimentally validated results in the presence of Exo III = 0.6 and 0.2 μM, respectively.(C) Time-dependent chemiluminescence changes of luminol generated by samples withdrawn at time intervals from the dynamic reaction module depicted in panel II of Figure 4: panel I, samples withdrawn at (a) t = 0 h, (b) t = 1 h, (c) t = 2 h; panel II, samples withdrawn at (d) t = 4 h, (e) t = 6 h, (f) t = 8 h, (g) t = 10 h; panel III, temporal, transient concentration changes of the (6)+(7)/(8) supramolecular structure generating chemiluminescence upon operation of the dynamic reaction module shown in Figure 4, panel II.Experimental conditions operating the dynamic process shown in panels I−III are A 2 -(6), 1 μM; B 2 -(7), 1 μM; T 2 -(8), 3 μM; Exo III, 0.4 μM.(D) Probing the effects of the concentrations of the fuel strand T 2 (panel I) and the concentrations of the Exo III (panel II) on the temporal transient generation of the (6)+(7)/(8) supramolecular structure and generation of chemiluminescence according to Figure 4, panel II.Part D, panel I: (a) Dotted points correspond to experimental data recorded at the conditions specified in (C); (a′) solid curves correspond to computationally simulated result using the kinetic model formulated in Figure S11, Supporting Information.(b′, c′) Computationally simulated transient concentrations of (6)+(7)/(8) supramolecular structure generating chemiluminescence at auxiliary conditions T 2 = 2 and 4 μM, respectively.(b, c) Dotted data correspond to experimentally validated results in the presence of T 2 = 2 and 4 μM, respectively.Part D, panel II: (a/a′) is the same condition with (a/a′) in panel I. (d′, e′) Computationally simulated transient concentrations of (6)+(7)/(8) supramolecular structure generating chemiluminescence at auxiliary conditions Exo III = 0.6 and 0.2 μM, respectively.(d, e) Dotted data correspond to experimentally validated results in the presence of Exo III = 0.6 and 0.2 μM, respectively.