Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

Abstract

The general design of the swinging arm nanostructure complex is shown in Fig. 1a, where a two-enzyme cascade consisting of glucose-6 phosphate dehydrogenase (G6pDH) 19 and malic dehydrogenase (MDH) 20 is displayed on a DNA double-crossover (DX) tile scaffold 21 (DNA sequences for all structures used in this study are shown in Supplementary Figs. S1-S7).G6pDH catalyzes the oxidation of glucose-6-phosphate and the reduction of NAD + to NADH.Subsequently, MDH catalyzes the reduction of oxaloacetate to malic acid using the NADH produced by G6pDH. To facilitate channeling of NADH between G6pDH and MDH, an NAD + -3 functionalized poly(T) 20 oligonucleotide was attached to the DNA tile surface halfway between G6pDH and MDH (see Supplementary Figs. S8-S21 for a detailed description of conjugation, assembly and purification of the nanostructured complex). Fig. 1b shows a native polyacrylamide gel electrophoresis (PAGE) analysis of the assembled enzyme complex, together with various sub-complexes. Both the gel results and the chromatogram resulting from sizeexclusion chromatography ( Supplementary Fig. S21) demonstrate assembly of the G6pDH-NAD + -MDH swinging arm cascade with >80% yield. The assembled mixture was further purified by size exclusion chromatography for enzyme activity assays. Assembly of the complete complex was also visualized by atomic force microscopy (AFM) ( Fig. 1c and Supplementary Fig. S57 for larger view images), where the presence of the enzymes on the structure is confirmed by differences in height ("brightness") compared to the surface of the DNA tile.To first explore the parameters and understand the kinetics and mechanism of the restricted diffusion mediated by the ssDNA swinging arm, we developed a simplified model system ( Supplementary Fig. S22). In this model, a Cy3 reporter dye takes the place of NAD + on the single-stranded poly(T) 20 arm, whereas a BHQ fluorescence quencher and a Cy5 energy transfer acceptor dye replace one or both enzymes on selected probe positions surrounding the swinging arm (Fig. 2a). An oligonucleotide sequence (5'-ATA GTG AAA) was extended from the 5' end of the poly(T) 20 sequence and positioned halfway between the quencher and acceptor, allowing the arm to transiently hybridize to the probes that each bear the complementary sequence (5'-TTT CAC TAT) in analogy to the binding of NAD + /NADH to the dehydrogenases. To characterize the distance dependence of the diffusive transport and binding mediated by the swinging arm using smFRET, we chose a design in which a single Cy5-labeled capture probe was placed at one of three topologically accessible distances from the Cy3-labeled arm: 7 nm (21 base pairs), 14 nm (42 base pairs) and 21 nm (63 base pairs). As shown in Fig. 2b, the most efficient hybridization to the capture probe was observed at 7 nm, where ~94% of all swinging arms were associated with the Cy5 probe at equilibrium (leading to high FRET). As the distance increased, the equilibrium fraction of captured swinging arms decreased to ~58% at 14 nm and only ...

DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices

“…They were able to control the enzymatic activity by opening and closing the tweezers using additional DNA oligonucleotides as fuel. Fu et al [58] fabricated multienzyme cascades on DNA templates, where substrate channeling was realized using a DNA strand as a flexible swinging arm between the immobilized enzymes. Linko et al [59] showed how modular DNA origami building blocks could be assembled together to form a tubular enzyme cascade nanoreactor ( Figure 2C) for detecting glucose.…”

Enhanced Activity of Immobilized or Chemically Modified Enzymes

“…Early in 1980s, Mansson and co-workers 87 immobilized the lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) on agarose beads with the aid of a bis-NAD analogue, followed by glutaraldehyde cross-linking to achieve an orientation of enzymes facing their active sites to each 24 Artificial swinging mediator for intermediate channeling.In the enzymatic reaction with cofactors involved, transportation of cofactors sometimes determines the overall activity. Very recently, Fu and colleagues78 presented an ingenious multi-enzyme-DNA complex by employing a swinging arm modified with NAD + on its terminal to mimic the substrate channeling (Figure 6). The swinging arm placed between the glucose-6-phosphate dehydrogenase (G6pDH) and malic dehydrogenase (MDH) facilitated the channeling of NADH/NAD + between the two enzymes.…”

Co‐immobilized Phosphorylated Cofactors and Enzymes as Self‐Sufficient Heterogeneous Biocatalysts for Chemical Processes

“…Inspired by nature, NAD(P)H and enzymes have been co‐immobilized on solid materials allowing their solid‐phase recycling and reusability for several operational cycles 3a, 4. These heterogeneous systems, however, have shown several drawbacks; the enzymatic activity towards the immobilized cofactors is low,5 the turnover numbers of the immobilized cofactors are poor (≤1),6 the re‐usability of both enzymes and cofactors is limited5, 7 and the fabrication of these systems is hardly scalable 8. However, the use of strong and non‐porous anionic exchangers has shown that the cofactor re‐utilization works in organic media9 but fails in aqueous media owing to the lixiviation of both NAD(P)H and enzymes from the matrix 10.…”