Vi

Figure 1 | Assembly of hexagon-like DNA strips and their structural imaging. a, Two-hexagon DNA strip assembly. b, Four-hexagon DNA strip assembly. c, AFM images of the two-hexagon strip: (I) large-scale image that includes a collection of strips, (II) image of a single strip, and (III) cross-sectional analysis of a single strip. d, AFM images of the four-hexagon strip: (IV) large-scale image that includes several strips, (V) image of a single strip, and (VI) cross-sectional analysis of a single strip.

proteins on the DNA template27. The width of the two-hexagon protein nanostructure is ~13 nm (after tip deconvolution), and that of the four-hexagon protein system ~33 nm (after tip deconvolution). Spatial control of FRET communication between the GOx and HRP in the two-and four-hexagon nanostructures is shown in Fig. 3b (images III and IV). The HRP was labelled with FAM and the GOx with TAMRA. Figure 3b (images III and IV) shows the fluorescence overlay images of the enzyme-two-hexagon DNA scaffold and of the enzyme-four-hexagon systems upon photoexcitation of FAM at Aex = 488 nm, respectively. In the two-hexagon protein hybrid system, the red fluorescence of TAMRA predominates, implying effective FRET, but in the four-hexagon protein hybrid structure the green fluorescence of FAM is intense, indicating less efficient FRET. These results confirm that the proteins associate with the DNA scaffolds, and that the energy transfer between the labelled proteins is controlled by the geometry of the DNA scaffolds.

Figure 3a shows the functional operation of the two-enzyme cascade on the two- and four-hexagon-like DNA scaffolds. The primary enzyme GOx biocatalyses the oxidation ofglucose to gluco-nic acid, with the concomitant formation of H2O2. The latter product acts as substrate for HRP, mediating the oxidation of -azino-bis[3-ethylbenzthiazoline-6-sulphonic-acid], ABTS2-, to the coloured product, ABTS", l ~ 414 nm. Different ratios [proteins:DNA primers] were tested, and a ratio of 1: 1 was found to be most efficient (see Supplementary Fig. S5). In Fig. 3c, curves I and II show the time-dependent formation of ABTS", using the two- and four-hexagon-like DNA scaffolds, respectively. For comparison, in Fig. 3c, curves III and IV depict the rates of ABTS" formation in the presence of the two enzymes GOx and HRP in the absence of the DNA scaffolds or in the presence of the foreign calf thymus DNA, respectively. Evidently, the enzyme cascade is not activated in the absence of the DNA scaffolds, or in the presence of a foreign DNA. However, the organization—and concentration—of the two proteins on the nucleic acid scaffolds activate the biocatalytic cascade. The generation of the H2O2 product in the vicinity of the secondary HRP biocatalyst allows the effective biocatalysed oxidation of ABTS2- owing to the high local concentration of the biocatalytically generated H2O2. Furthermore, we demonstrate the spatial control of the biocatalytic cascade by anchoring it to the DNA scaffolds. The oxidation of ABTS2- is ~1.2-fold higher in the presence of the two-hexagon structure, as compared to the four-hexagon system. This is explained by the larger distances separating the GOx/HRP couple in the four-hexagon structure, which leads to lower local concentrations of H2O2 at the HRP site, due to partial diffusion of H2O2 to the bulk solution. It should be noted that the difference of ~20% in the rate of the biocatalytic cascades operating on the two-and four-hexagon templates is very reproducible, and the kinetic curves were well statistically resolved by five sets of experiments performed on each template. Furthermore, the kinetic analysis of the biocatalytic cascades operating on the templates follow the Michaelis-Menten model (see Supplementary Fig. S6, panels I to IV). The observed distance dependence of the biocatalytic cascades operating on the two- and four-hexagon templates is rationalized in terms of the flux of the H2O2 transported to the HRP through a cross-section, being part of the spherical surface area controlled by the distance separating the two enzymes (see Supplementary Fig. S6 and explanation). It should be noted that, in principle, the intermolecular single hexagon-type structures could be designed

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