DNA walking machines. Tuberfield review, Nature Nanotechnology, vol 2, may 2007, p.275. NANOANDES 2017, November 22-29, Buenos Aires, Argentina 76

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1 DNA walking machines A* B A*B A B* Tuberfield review, Nature Nanotechnology, vol 2, may 2007, p.275 NANANDES 2017, November 22-29, Buenos Aires, Argentina 76

2 NANANDES 2017, November 22-29, Buenos Aires, Argentina 77

3 DNA-based multienzyme catalysts Enzyme cascades activated on topologically programmed DNA scaffolds TTERS fer. Wilner, Yossi Weizmann, Ron Gill, leg Lioubashevski, Ronit Freeman and tamar Willner* The ability of DNA to self-assemble into one-, two- and three-dimensional nanostructures 1 14, combined with the precision that is now possible when positioning nanoparticles or proteins on 2 DNA scaffolds, provide a promising approach for the self-organization 2 Glucose of composite nanostructures Predicting and controlling the functions that emerge in self-organizedbiomolecular 2 2 ABTS nanostructures 2- is a major challenge in systems biology, and although a number of innovative examples Gx Gluconic have been reported 28 30, the emergent properties of systems in which RP enzymes are coupled together acid S have not been fully explored. ere, we report the self-assembly of a DNA scaffold made of DNA strips that ABTS include hinges to which biomolecules can be tethered. We attach either two enzymes or a cofactor enzyme pair to the scaffold, and show that enzyme cascades or cofactor-mediated biocatalysis can proceed effectively; similar processes are not observed in diffusion-controlled homogeneous 2 mixtures 2 of 2 the same components. Furthermore, because the relative position 2 Glucose 2 of the 2 2 two enzymes or the cofactor enzyme pair is determined 2 by 2 the topology of the DNA scaffold, 2 2 it is possible to control the Gluconic reactivity of the system through the design of the individual Gx be detected. Figure 1c, image, shows an AFM image of a single two-hexagon strip, and image presents the cross-sectional analysis of the strip. The height of the DNA strip corresponds to 2 nm and the width 25 nm. This height is consistent with the reported 0.08 values for duplex DNA structures on surfaces 27 (for the discussion on the width parameter, see below). Figure 1d shows AFM images 4 of the strips resulting from the self-assembly 0.06 of the four nucleic 3.5 acids 3 to 6. Bundles and single strips are also observed (Fig. 1d, ABTS 2-3 = 2.5 image V). mages V and V in Fig. 1d depict a single fourhexagon strip and N the cross-sectional analysis 0.04 of the strip, respect ively. S TheNwidth of the single strip corresponds to 45 nm. This 1 value should N be S compared S with the width of the two-hexagon 0.5 N 0.02 strips of Fig. 1c, image, measured as 25 nm. Taking into nm V38.5 m account the tip dimensions, these values translate to width values of 13 nm for the two-hexagon strips and0 33 nm for the fourhexagon strips. These values are in full agreement 0 with the estimated x (nm) geometric widths of the strips (see Supplementary Fig. S1). The formation of the DNA strips was further supported by scanning elec- Time (s) tron 2 microscopy ABTS 2- (SEM) analyses (see Supplementary Fig. S2). 3.5 The formation of these self-organized Time-dependent DNA strips, and the possibility of controlling physicochemical properties of chemical com- 2.5 absorbance 3 changes DNA strips. This method could lead to the self-organizationrp as a result of the oxidation of ABTS2- by acid ponents attached to the DNA scaffolds, were demonstrated by 2 of complex multi-enzyme cascades. fluorescence resonance energy transfer the Gx RP (FRET) experiments. cascade 1.5 in the presence The self-assembled DNA nanostructures consist of a set of The dyes ABTS 5-carboxyfluorescein N-succinimidyl ester (FAM) and 1 0 single-stranded nucleic acids of pre-designed sequence composition 5-carboxy-tetramethylrhodamine N-succinimidyl of () the ester two-hexagon (TAMRA) scaffold, () the 0.5 that show partial complementarities to form hexagon-like hybridizations that lead to the formation of the strips. Figure 1a depicts the were used to functionalize the nucleic four-hexagon acids 7 and 8, which scaffold, are () in the complementary to the free nucleic acid470 tethers nm at the edges of the 7 m schematic self-organization of the strips by the interaction of two two-hexagon (nucleic acids 1 and absence 2) and four-hexagon of any strips DNA, and (V) in the x (nm) nucleic acids, 1 and 2, which have partial base complementarity. (nucleic acids 3 and 6). presence of foreign calf 0.02 thymus DNA Each of the single-stranded nucleic acids includes 70 bases, and We found by fluorescence measurements in solution and confocal fluorescence microscopy analyses on surfaces that FRET between the formation of the strips includes, in each hexagon, 60 bases 0.03 and a free apendant nucleic acid composed of 10 bases linked to the FAM and TAMRA dyes occurred, and that the efficiency of the the hexagons. These tethers are able to act as hinges for the association of the biomolecules with the DNA scaffold. Figure 1b shows Supplementary Figs S3 and S4). Glucose 0.04 MB + FRET was controlled by the distance separating the dyes LETTERS (seemb the schematic 0.08 self-assembly of strips NANANDES consisting of four PUBLSED 2017, hexagon November construction units. Four single-stranded nucleic acids, 3, 4, 5 and 6, were, then, used as scaffolds for the self-assembly of two concate-nad 0.05 NLNE: 22-29, The resulting Buenos 29 two-hexagon MARC Aires, Argentina 2009 and four-hexagon D: /NNAN nanostructures 78 were used; 3 and 6 consisted of 100 bases each, and 4 and 5 nated enzymes, glucose oxidase (Gx) and horseradish peroxidase GD + NAD Absorbance LETTERS PUBLSED NLNE: 29 MARC 2009 D: /NNAN NATURE NANTECNLGY D: /NN z (nm) z (nm) Absorbance

4 NANANDES 2017, November 22-29, Buenos Aires, Argentina 79

5 DNA and micro-nano(electronics) prepared with Raluca Tiron, CEA researcher NANANDES 2017, November 22-29, Buenos Aires, Argentina 80

Catalytic Nucleic Acids (DNAzymes) as Functional Units for Logic Gates and Computing Circuits: From Basic Principles to Practical Applications

Catalytic Nucleic Acids (DNAzymes) as Functional Units for Logic Gates and Computing Circuits: From Basic Principles to Practical Applications Journal: Manuscript ID: CC-FEA-12-2014-009874.R1 Article Type: