Dynamic Catalysis Guided by Nucleic Acid Networks and DNA Nanostructures

Abstract

Nucleic acid networks conjugated to native enzymes and supramolecular DNA nanostructures modified with enzymes or DNAzymes act as functional reaction modules for guiding dynamic catalytic transformations. These systems are exemplified with the assembly of constitutional dynamic networks (CDNs) composed of nucleic acid-functionalized enzymes, as constituents, undergoing triggered structural reconfiguration, leading to dynamically switched biocatalytic cascades. By coupling two nucleic acid/enzyme networks, the intercommunicated feedback-driven dynamic biocatalytic operation of the system is demonstrated. In addition, the tailoring of a nucleic acid/enzyme reaction network driving a dissipative, transient, biocatalytic cascade is introduced as a model system for out-of-equilibrium dynamically modulated biocatalytic transformation in nature. Also, supramolecular nucleic acid machines or DNA nanostructures, modified with DNAzyme or enzyme constituents, act as functional reaction modules driving temporal dynamic catalysis. The design of dynamic supramolecular machines is exemplified with the introduction of an interlocked two-ring catenane device that is dynamically reversibly switched between two states operating two different DNAzymes, and with the tailoring of a DNA-tweezers device functionalized with enzyme/DNAzyme constituents that guides the dynamic ON/OFF operation of a biocatalytic cascade by opening and closing the molecular device. In addition, DNA origami nanostructures provide functional scaffolds for the programmed positioning of enzymes or DNAzyme for the switchable operation of catalytic transformations. This is introduced by the tailored functionalization of the edges of origami tiles with nucleic acids guiding the switchable formation of DNAzyme catalysts through the dimerization/separation of the tiles. In addition, the programmed deposition of two-enzyme/cofactor constituents on the origami raft allowed the dynamic photochemical activation of the cofactor-mediated biocatalytic cascade on the spatially biocatalytic assembly on the scaffold. Furthermore, photoinduced "mechanical" switchable and reversible unlocking and closing of nanoholes in the origami frameworks allow the "ON" and "OFF" operation of DNAzyme units in the nanoholes, confined environments. The future challenges and potential applications of dynamic nucleic acid/enzyme and DNAzyme conjugates are discussed in the conclusion paragraph.

Figure 1

(A) Schematic assembly of a constitutional dynamic network (CDN) composed of four equilibrated constituents, CDN X, being dynamically reconfigured by auxiliary triggers. The constituents are composed of four components A, A′, B, B′, yielding the equilibrated constituents AA′, BB′, AB′, and BA′. The T₁-triggered stabilization of constituent AA′ in CDN X leads to the dynamic reconfiguration of CDN X into the re-equilibrated CDN Y where AA′ is enriched at the expense of AB′ and BA′, resulting the concomitant enrichment of BB′ in the equilibrated mixture. Subjecting CDN X to trigger T₂ stabilizes the constituent AB′ and as a result leads to the dynamic reconfiguration of CDN X to Z, where constituents AB′ and BA′ are enriched and constituents AA′ and BB′ are downregulated in their content in the resulting and BB′ re-equilibrated network. Adapted from ref (65) with permission. Copyright 2020, American Chemical Society. (B) Examples of reversible triggers that guide the programmed dynamic reconfiguration of CDNs.

Figure 2

(A) Schematic nucleic acid-triggered operation of constitutional dynamic networks, CDNs, controlling the biocatalytic cascade consisting of GOx/HRP (see inset panel I). (B) Synthesis of enzymes functionalized with single nucleic acid tether A-GOx and A′-HRP. (C) Contents of the equilibrated constituents in CDN “L”, CDN “M”, and CDN “N”, evaluated by the activities of the Mg²⁺-ion-dependent DNAzymes associated with the constituents. (D) Time-dependent absorbance changes of ABTS^•-, reflecting the efficacy of the GOx/HRP biocatalytic cascade associated with CDN “L”, panel I, curve (i), and T₁-triggered reconfigured CDN “M”, panel I, curve (ii); Time-dependent absorbance changes of ABTS^•- reflecting the efficacy of the GOx/HRP biocatalytic cascade associated with CDN “L”, panel II, curve (i), and T₂-triggered reconfigured CDN “N”, panel II, curve (ii). (E) Panel I-Cyclic operation of the GOx/HRP biocatalytic cascade upon the reversible switching, between CDN “L” and CDN “M” using the triggers T₁ and T₁′, and panel II-Cyclic operation of the GOx/HRP biocatalytic cascade upon the reversible switching, between CDN “L” and CDN “N” using the triggers T₂ and T₂′. Figure adapted from ref (101) with permission. Copyright 2020 Nature Publishing Group.

Figure 3

Orthogonal coupled operation of two constitutional dynamic networks that guide the dynamic cascaded biocatalytic reactions consisting of GOx/HRP and ADH/NAD⁺. The two CDNs “L” and “P” are subjected to two hairpins H_aa′ and H_dd′ that fuel the coupled operation of the networks. Cleavage of H_aa′ by constituent CC′ associated with CDN “P” yields fragment H_aa-1 that stabilizes constituent AA′ in CDN L resulting in the temporal dynamic enrichment of AA′ and the concomitant enrichment of constituent BB′. Simultaneously, cleavage of hairpin H_dd′ by constituent BB′ associated with CDN L yields strand H_dd-1′ that stabilized constituent DD′ of CDN P and the concomitant dynamic enrichment of constituent CC′. The operation of the two networks leads to the dynamic temporal enrichment of AA′ associated with CDN L and to the accompanying temporal feedback-driven enhancement of the GOx/HRP cascade (Figure 3A, panel I), and the concomitant temporal feedback-driven enrichment of constituent DD′ of CDN P accompanied by the temporal enhancement of the ADH/NAD⁺ cascade followed by the reduction of Methylene Blue, MB⁺, to reduced Methylene Blue, MBH (Figure 4, panel II). (B) Panel I: Time-dependent rates of ABTS^•- formation by temporal samples of the reaction mixture generated by the reaction mixture. Panel II: Catalytic temporal rates of ABTS^•- formation by the dynamically feedback-driven coupled CDNs system. (C) Panel I: Time-dependent rates of MBH formation by temporal samples of the reaction mixture generated by the ADH/NAD⁺ cascade. Panel II: Catalytic temporal rates of MBH formation by the dynamically feedback-driven coupled CDNs system. Figure adapted from ref (101) with permission. Copyright 2020 Nature Publishing Group.

Figure 4

Assembly and operation of an artificial photosynthetic system driven by constitutional dynamic networks: (A) Composition and triggered reconfiguration of the artificial photosynthetic constitutional dynamic network. The artificial photosynthetic process corresponding to the photosensitized generation of the bipyridines radical cation, V^+•, and the subsequent FNR catalyzed reduction of NADP⁺ to NADPH are displayed in panel I. The reversible reconfiguration processes of the CDNs are stimulated by triggers T₁/T₁′ or T₂/T₂′ generation or separation of triplex domains in the constituents, panel II; the contents of the constituents in the different CDNs are transduced by the Mg²⁺-ion-dependent DNAzyme reported units associated with the constituents, panel III. (B) Concentration of the constituents in CDNs, Z, Z_a, and Z_b transduced by the Mg²⁺-ion-dependent DNAzyme reported units. (C) Absorption spectra corresponding to the V^+• generated by the equilibrated reconfigured CDNs: (i) CDN Z, (ii) CDN Z_a, (iii) CDN Z_b. (D) Cyclic and reversible CDNs-guided operation of the artificial photosynthetic network monitored by the light-generated V^+•. (E) Time-dependent concentrations of NADPH generated by the artificial photosynthetic cascades guided by CDNs Z, Z_a, and Z_b. Figure adapted from ref (102) with permission (Creative Commons, CC BY 4.0).

Figure 5

(A) Schematic operation of a nucleic acid-driven dynamic, dissipative, transient glucose oxidase (GOx)/horseradish (HRP) biocatalytic cascade. The bocatalytic cascade involves the aerobic GOx catalyzed oxidation of glucose to gluconic acid and H₂O₂ and the subsequent HRP-catalyzed oxidation of ABTS^2– to ABTS^•-, Inset X, or the aerobic GOx catalyzed oxidation of glucose to gluconic acid and H₂O₂ and the cascaded HRP catalyzed generation of chemiluminescence in the presence of luminol/H₂O₂, Inset Y. (B) Time-dependent transient rates corresponding to the oxidation of ABTS^2– to ABTS^•- by the GOx/HRP biocatalytic cascade associated with temporal samples withdrawn from the dynamic, transient, reaction module shown in (A). Panel I: Samples corresponding to the temporal buildup of the transient T₁/A₁-GOx + A₂-HRP. Panel II: Samples corresponding to the temporal depletion of the T₁/A₁-GOx + A₂-HRP intermediate. Panel III: Transient dissipative absorbance changes of ABTS^•- associated with the temporal build-up and depletion of the biocatalytic intermediate T₁/A₁-GOx + A₂-HRP. (C) Panel I: Chemiluminescence spectra generated by the GOx/HRP biocatalytic cascade in the presence of luminol/H₂O₂, using samples withdrawn at time intervals from the dissipative reaction module shown in (A). Panel II: Temporal chemiluminescence intensities generated upon the transient build-up and depletion of the intermediate T₁/A₁-GOx + A₂-HRP bicatalytic supramolecular complex. Figure adapted from ref (103) with permission (Creative Commons, CC BY-NC).

Figure 6

(A) Dynamic reversible and cyclic reconfiguration of an interlocked DNA two-ring catenane system using fuel/antifuel strands as triggers. The reconfiguration of the catenane in state A to state B yields a hemin/G-quadruplex DNAzyme unit in ring α1 that stimulates the hemin/G-quadruplex catalyzed generation of chemiluminescence in the presence of luminol/H₂O₂ or the catalyzed oxidation of ABTS^2– by H₂O₂ to form the colored ABTS^•-. (B) Chemiluminescence spectra generated by curves (a) and (c)—state A, curves (b) and (d)—state B, and inset—cyclic chemiluminescence responses of the system. (C) Switchable absorbance chances of ABTS^•- generated upon the cyclic reconfiguration of the catenane between states B and A. At times marked (a), the system is switched to state A, and at times marked (b), the molecular device is switched to state B. (D) Dynamic reversible reconfiguration and switching of an interlocked two-ring catenane between state X and state Y, using fuel/antifuel strand displacement triggers, that activate switchable Mg²⁺-ion-dependent DNAzyme and Zn²⁺-ion-dependent DNAzyme functions. (E) (i) Switchable catalytic activities of the Zn²⁺-ion-dependent DNAzyme; (ii) switchable catalytic activities of the Mg²⁺-ion-dependent DNAzyme. At times marked with arrows (a), L₃′ is added to yield state X, and at times marked with (b), L₃ is added to yield state Y. Figure adapted from ref (112) with permission. Copyright 2015, American Chemical Society.

Figure 7

(A) Schematic operation of a tweezers-like biocatalytic conjugate consisting of three biocatalysts β-Gal/GOx/hemin-G-quadruplex using 18-crown-6-ether and K⁺-ions as reconfiguration triggers. The biocatalytic cascade involves the β-Gal catalyzed hydrolysis of lactose to yield galactose and glucose, the subsequent GOx-catalyzed aerobic oxidation of glucose to gluconic acid and H₂O₂, and the subsequent hemin/G-quadruplex-catalyzed oxidation of ABTS^2– by H₂O₂ to the colored ABTS^•- product. The “closed” state of the tweezers, state G, is reconfigured through separation of the G-quadruplex by 18-crown-6-ether, into the “open” tweezer configuration, state H. The “open” tweezers, state H, is reconfigured into state G upon addition of K⁺-ions that bridges the tweezers arm by a K⁺-ion stabilized hemin/G-quadruplex stabilized unit. The tweezers arms are modified with a fluorophore (Cy 5.5)/quencher (Iowa Black RQ) pair that transduces the mechanical opening/closing of the tweezers by the fluorescence response of the fluorophore/quencher pair. (B) Switchable fluorescence changes upon the cyclic “mechanical” opening and closing of the tweezers, in the presence of added crown-ether/K⁺-ions, respectively. (C) Time-dependent absorbance change of ABTS^•- upon the cyclic “ON” and “OFF” activation of the three-catalyst β-Gal/GOx/hemin-G-quadruplex cascade. At times marked with (a), the K⁺-ion-stabilized β-Gal/GOx/hemin-G-quadruplex “closed” tweezers structure, state G, is formed, and at times marked with (b), the crown-ether stimulated opening of the tweezers proceeds to yield the switched “OFF” biocatalytic cascade, state H. (D) Dynamic switching of the GOx/HRP biocatalytic cascade using a double-cross over DNA structure locked by an immobile four-way junction. The device is switched between “open” and “closed” states by applying fuel/antifuel strands. (E) Time-dependent absorbance changes of ABTS^•- generated by the GOx/HRP bienzyme cascade in the presence of (a) “open” tweezer structure; (b) “closed” tweezer structure. Inset: Reversible operation of the GOx/HRP cascade by the tweezers device. Figure 7A–C adapted from ref (113) with permission. Copyright 2014 John Wiley and Sons. Figure 7D and E adapted from ref (117) with permission. Copyright 2014 John Wiley and Sons.

Figure 8

(A) Schematically switchable dimerization and separation of two origami tiles using hemin/G-quadruplex DNAzyme bridges, triggered by K⁺-ions and 18-crown-6-ether. Inset: DNAzyme catalyzes the Amplex-Red oxidation of by H₂O₂ to the fluorescent Resorufin. (B) AFM images and cross section analysis corresponding to the dimerized, panel I; K⁺-ion-separated tiles, panel II; and crown-ether redimerized tiles, panel III. (C) Switchable catalyzed activation of the hemin/G-quadruplex DNAzyme upon the triggered separation and dimerization of the origami tiles. Figure adapted from ref (121) with permission. Copyright 2018 American Chemical Society.

Figure 9

(A) Schematic photoinduced activation of a NAD⁺-cofactor biocatalytic cascade consisting of G6pDH and lactate dehydrogenase (LDH), on a DNA origami tile, using a mediating NAD⁺-cofactor tethered to an azobenzene photoisomerizable foothold associated with the origami tile. (B) Switchable ON/OFF catalytic activities of the photoresponsive NAD⁺-mediated bienzyme cascade: Switching “OFF” in the presence of the trans-azobenzene modified foothold (generated upon visible light illumination), switching “ON” in the presence of the cis-azobenzene foothold (generated upon UV light illumination). Figure adapted from ref (122) with permission. Copyright 2018 American Chemical Society.

Figure 10

(A) Dynamic control over the glucose oxidase (GOx)/horseradish peroxidase (HRP) cascade by the rotatable transitions of the biocatalytic cascade within an origami framework using the strand displacement principle. (B) Clockwise dynamic transitions of the GOx/HRP cascade across the states P₁, P₂, and P₃. The biocatalytic cascade involves the GOx catalyzed aerobic oxidation of glucose to gluconic acid and H₂O₂, followed by the HRP catalyzed oxidation of ABTS^2– by H₂O₂ to the colored ABTS^•- product. (C) Time-dependent absorbance changes corresponding to the GOx/HRP biocatalytic cascade generating ABTS^•- by the states P₁, P₂, and P₃. Figure adapted from ref (123) with permission. Copyright 2019 John Wiley and Sons.

Figure 11

(A) Schematic photoinduced dynamic unlocking and locking of a nanohole in an origami raft using photoisomerizable trans/cis-azobenzene-modified nucleic acid locks. (B) AFM images corresponding to panel I: the locked nanohole domain associated with the DNA origami rafts; panel II: the photoinduced unlocked nanohole-modified nanohole rafts. (C) Light-induced switchable closed/open nanohole-modified origami rafts. (D) Schematic photoresponsive DNA origami tile that includes a trans-azobenzene-locked domain functionalized on opposite faces with engineered G-quadruplex subunit tethers. The photoinduced unlocking of the trans-azobenzene locks yields nanoholes that, in the presence of K⁺-ions and hemin, enable the self-assembly of hemin/G-quadruplex DNAzyme units in the confined nanohole environment. The DNAzyme catalyzes the Amplex Red oxidation of by H₂O₂ to the fluorescent Resorufin. By the cyclic cis/trans photoisomerization of locking units, the reversible opening and closing of the nanoholes proceeds, resulting in the switchable ON/OFF biocatalytic functions in the nanohole domain. (E) Switchable ON/OFF hemin/G-quadruplex catalyzed oxidation of Amplex Red by H₂O₂ to form fluorescent Resorufin. Figure adapted from ref (124) with permission (Creative Commons, CC BY 4.0).

Figure 12

(A) Schematic reaction module consisting of an equilibrated constitutional dynamic network, CDN T, two functional duplexes L₁/T₁ and L₂/T₂ and nicking enzyme, Nt.BbvCI, that undergoes the L₁′- or L₂′-fueled transient transition of CDN T to CDN Q or CDN R and to the dissipative nickase-driven recovery to CDN T. The transient transition of CDN T to Q leads to the emergence of the transient active Mg²⁺-ion-dependent DNAzyme (function 1), whereas the L₂′-stimulated dynamic transition of CDN T to CDN R yields the emergent transient hemin/G-quadruplex DNAzyme activity (function 2), panel III. (B) Transient catalytic rates of the emergent Mg²⁺-ion-dependent DNAzyme (function 1): upon L₁′-triggered the temporal transient transition of CDN T to CDN Q, curve (i); upon L₂′-triggered the temporal reconfiguration of CDN T to CDN R and back, curve (ii). (C) Transient catalytic rates of the emergent hemin/G-quadruplex DNAzyme (function 2): upon L₂′-triggered the temporal transient transition of CDN T to CDN R and back, curve (iii); upon L₁′ triggered the temporal reconfiguration of CDN T to CDN Q and back, curve (iv). Adapted from ref (126) with permission. Copyright 2020, American Chemical Society.