-SUPPORTING INFORMATION-

Transcription

1 -SUPPORTING INFORMATION- Spatially Organized Enzymes Drive Cofactor-Coupled Cascade Reactions Tien Anh Ngo, Eiji Nakata, Masayuki Saimura, and Takashi Morii* Contribution from Institute of Advanced Energy, Kyoto University, Uji, Kyoto , Japan. * To whom correspondence should be addressed: Prof. Takashi Morii Tel.: Fax: t-morii@iae.kyoto-u.ac.jp S1

2 Table of contents Figure S1. Molecular models of ZS-XR and G-XDH... S5 Figure S2. SDS-PAGE analysis of purified ZS-XR... S6 Figure S3. Enzymatic activities of ZS-XR and a mutant XR... S7 Table S1. Kinetic parameters for a mutant XR and ZS-XR for the oxidation of NADH... S8 Figure S4. Illustrations show the structures of DNA origami scaffolds used in this study... S9 Note S1. Estimating the concentration of DNA origami and DNA origami-protein-complexes... S10 Figure S5. Gel electrophoretic and AFM analyses of the DNA origami scaffold (NB)... S10 Table S2. Total numbers and occupancies of the DNA scaffolds assembled with ZS-XR or G-XDH analyzed by AFM... S11 Note S2. Estimating the actual number of ZS-XR and G-XDH molecules bound to the specific binding sites on the DNA scaffold by volume analyses... S12 Figure S6. Determination of the actual number of ZS-XR molecules on the DNA scaffold... S14 Figure S7. Determination of the actual number of G-XDH molecules on the DNA scaffold... S15 Figure S8. Estimating the actual number of ZS-XR molecules bound on I-4XR... S16 Figure S9. Effect of the DNA scaffold on the catalytic activity of ZS-XR... S17 Figure S10. Effective removal of the unbound ZS-XR upon purification of the ZS-XR bound DNA scaffold... S18 Figure S11. Illustrations of coassembled ZS-XR and G-XDH on the DNA scaffolds with various interenzyme distances... S20 Figure S12. Specific binding ability and the actual number of enzymes inside cavity I of the DNA scaffold (I-4XR/4XDH)... S21 Figure S13. Specific binding ability and the actual number of enzymes inside the cavities of the DNA scaffold (I-4XR/II-4XDH)... S23 Figure S14. Specific binding ability and the actual number of enzymes inside cavities I and III of the DNA scaffold (I-4XR/III-4XDH)... S25 S2

3 Note S3. Calculation of the interenzyme distance between ZS-XR and G-XDH in the solution... S27 Figure S15. Time-course profiles of the change of absorbance at 340 nm in the enzyme cascade reactions with various ZS-XR/G-XDH interenzyme distances for bimolecular diffusion... S28 Figure S16. Analyses of the metabolites formation by the enzyme cascade reactions for bimolecular diffusion by HPLC... S29 Table S3. Quantitation of the metabolites by HPLC... S30 Figure S17. Distance dependency of the enzyme cascade reactions on the DNA scaffold for unimolecular (NAD + ) diffusion... S31 Note S4. Normalization of the initial rate of NADH regeneration (V ini) by G-XDH and the amount of products on the DNA scaffold... S32 Table S4. Reaction parameters for the enzyme cascade in bimolecular transport system... S33 Table S5. Reaction parameters for the enzyme cascade in unimolecular transport system... S33 Note S5. Xylitol or NAD + diffusion model between ZS-XR and G-XDH... S34 Figure S18. Model of xylitol or NAD + diffusion in a single ZS-XR/G-XDH pair... S35 Note S6. Concentration dependency for the efficiency of cascade reaction by coassembled enzymes on the DNA scaffold... S36 Figure S19. Comparison of the reaction efficiency of ZS-XR/G-XDH on the DNA scaffold and in bulk solution at various concentrations of enzymes... S37 Table S6. Parameters for the enzyme cascade reactions with varying concentrations of enzymes/dna scaffold in the unimolecular transport system... S39 Table S7. Michaelis constants of ZS-XR... S40 Table S8. Michaelis constants of G-XDH... S40 Table S9. Nucleotide sequences of primer pairs for construction of an expression vector for ZS-XR... S41 Table S10. Unmodified staple strands used for the assembly of the DNA origami scaffolds... S42 Table S11. Modified nucleotide sequences for the staple strands containing the binding sites for G-XDH used for preparation of DNA scaffolds... S46 Table S12. Modified nucleotide sequences for the staple strands containing the binding sites for ZS-XR S3

4 with BG modification used for preparation of DNA scaffolds... S47 References... S48 S4

5 Figure S1. Molecular models of ZS-XR and G-XDH. (a) A molecular model for the complex of ZS-XR and a hairpin DNA constructed by the crystal structures of the DNA complex of zif268 (PDB ID: 1ZAA), the complex of BG and SNAP-tag (PDB ID: 3KZY), and XR (PDB ID: 3TJL). (b) A molecular model for the complex of G-XDH and a hairpin DNA constructed by the crystal structures of XDH (PDB ID: 1ZEM) and the DNA complex of GCN4 (PDB ID: 1DGC). Molecular models were constructed by using Discovery Studio (version 3.1, Accelrys Inc.). S5

6 Figure S2. (a) SDS-PAGE analysis of purified ZS-XR. Lane M: molecular weight ladder (M.W ), lane 1: ZS-XR after purification. (b) An amino acid sequence of ZS-XR. S6

7 Figure S3. Enzymatic activities of ZS-XR and a mutant XR. (a) Time-course plots for the oxidation of NADH (0.15 mm) by ZS-XR (55 nm; solid circle) or a mutant XR (55 nm; open circle) in the presence of xylose (200 mm) were monitored by absorbance at 340 nm. (b) Catalytic activities of ZS-XR and a mutant XR. One unit is defined as the amount of enzyme that consumes 1 µmol NADH per minute. Error bars were generated as the standard deviation of the mean from at least three replicates. S7

8 Table S1. Kinetic parameters for a mutant XR and ZS-XR for the oxidation of NADH. Parameters Enzyme mutant XR ZS-XR K m (M) (14.9 ± 4.6) 10-5 (7 ± 2.4) 10-5 k cat (s -1 ) 883 ± ± 178 k cat/k m (M -1 s -1 ) (6 ± 0.7) 10 6 (11 ± 0.8) 10 6 V max (M s -1 ) (4.9 ± 1) 10-5 (4.2 ± 1) 10-5 [mutant XR] = [ZS-XR] = 55 nm, [NADH] = µm, [xylose] = 200 mm in a buffer (ph 7.6) containing 40 mm Tris-HCl, 20 mm acetic acid, 100 mm NaCl, 12.5 mm MgCl 2, 0.02% Tween-20, and 1 µm ZnCl 2 at 25 C. S8

9 Figure S4. Illustrations show the structures of DNA origami scaffolds used in this study. (a) Illustration of the designed DNA scaffold with three cavities. (b) The DNA scaffolds used in this study. The stem loops in red and blue denote the binding site for ZS-XR with the BG modification and the binding sites for G-XDH, respectively. S9

10 Note S1. Estimating the concentration of DNA origami and DNA origami-protein complexes. DNA origami (10 nm) was prepared as described in Materials and Methods. To determine the molar absorbance coefficient of DNA origami, the recover yield of DNA origami after gel filtration was obtained by quantitation of the agarose gel electrophoretic band intensity (90 ± 5%, Figure S5). From the absorption spectrum of purified DNA origami (9 nm) measured by Nanodrop (Thermo Fisher Scientific Inc.), the molar absorbance coefficient of DNA origami at 260 nm was determined to be (5.6 ± 0.5) 10 7 M -1 cm -1. The molar absorbance coefficients of ZS-XR and G-XDH (monomer) at 260 nm, which were determined to be (5.4 ± 0.1) 10 4 M -1 cm -1 and (1.1 ± 0.1) 10 4 M -1 cm -1, respectively, were negligible to the value of DNA origami alone. Therefore, the concentration of DNA origami-protein complex after purification was determined by using the molar absorbance coefficient of DNA origami. (a) M (b) 100 nm Figure S5. Gel electrophoretic and AFM analyses of the DNA origami scaffold (NB). (a) Agarose gel electrophoretic analysis of the DNA scaffold before and after the purification by gel filtration (400 µl volume of Sephacryl S-400 in Ultrafree-MC-DV with a buffer, ph 7.0). The gel was stained with ethidium bromide (EtBr) and visualized by using Molecular Imager FX pro (BioRad) under EtBr-mode. Lane M: 1000 bp DNA ladder; lane 1: M13mp18 single-stranded DNA alone (10 nm); lane 2: DNA origami scaffold before the purification; lane 3: DNA origami scaffold after the purification. The black arrow indicated the DNA scaffold and the blue arrow show the excess staple strands. (b) AFM analysis of the DNA scaffold after the purification. S10

11 Table S2. Total numbers and occupancies of the DNA scaffolds assembled with ZS-XR or G-XDH analyzed by AFM. Adaptor fused enzymes DNA scaffolds Numbers of the expected structure (N total) Numbers and occupancies of the modified DNA bound at the expected position scaffold (N expected posi) [occupancy %] bound at unexpected positions (N unexpected posi) [occupancy %] ZS-XR a I-4XR/II-4XDH [95%] 42 [6%] I-4XR [97%] 49 [4%] G-XDH b I-4XR/II-4XDH [78%] 37 [7%] a: [DNA scaffold] = 13.6 nm, [ZS-XR] = 300 nm. Reactions were carried out for 30 min on ice. b: [DNA scaffold] = 13.6 nm, [G-XDH] = 300 nm. Reactions were carried out for 30 min on ice. S11

12 Note S2. Estimating the actual number of ZS-XR and G-XDH molecules bound to the specific binding sites on the DNA scaffold by volume analyses. In order to evaluate the actual number of ZS-XR molecules bound to the predesigned binding sites on the DNA scaffold, the volume of ZS-XR in each cavity of DNA scaffold (4-2-1-XR), which was designed to have four binding sites for ZS-XR inside cavity I, two in cavity II, and one in cavity III (Figure S6a), were determined using volume analyses of AFM images and listed as frequency distribution of molecular volumes of ZS-XR (Figure S6b). The average volume of ZS-XR in each cavity of XR was determined as 204 ± 40 nm 3 (cavity III), 363 ± 98 nm 3 (cavity II), and 778 ± 115 nm 3 (cavity I), which corresponded to the volume occupied by one, two and four molecules of ZS-XR at the specific binding sites on the DNA scaffold. Standard curve for the number of ZS-XR molecules versus molecular volumes was determined as shown in Figure S6c. In the case of G-XDH, the volume of G-XDH in each cavity of the DNA scaffold (4-2-1-XDH), which was designed to have four binding sites for G-XDH inside cavity I, two in cavity II, and one in cavity III (Figure S7a), were determined using volume analyses of AFM images and listed as frequency distribution of molecular volumes of G-XDH (Figure S7b). In cavity III, unique distribution corresponding to one G-XDH dimer was observed (255 ± 47 nm 3 ). In contrast, frequency distributions covered a broader range of volumes in the case of cavities II and I, indicating a distribution from one to four G-XDH dimers in the cavity. In cavity II, the major fraction (58%) was centered at 492 ± 70 nm 3, which corresponded well to the volume of two G-XDH dimers. In cavity I, the major fraction (50%) was centered at 899 ± 113 nm 3, which corresponded well to the volume of four G-XDH dimers. Standard curve for the number of dimeric G-XDH molecules versus molecular volume was obtained as shown in Figure S7c. These standard curves (Figure S6 and S7) were used to determine the actual number of ZS-XR molecules and G-XDH molecules (in the dimeric form) bound to the specific DNA sequences on the DNA scaffold. 1: The actual number of bound ZS-XR or G-XDH was determined by using the following equation S12

13 where n actual is the actual number of ZS-XR or G-XDH in the cavity, P specific is the yield of assembled ZS- XR or G-XDH on the DNA scaffold (see Materials and Methods), and F k (k =1, 2, 3, or 4) is the population of each number of ZS-XR molecules. In the case of G-XDH, F k (k = 2, 4, 6, or 8) were used for its homodimer configuration. To analyze coassembled ZS-XR and G-XDH on the DNA scaffold, the actual number of ZS-XR in the cavity was determined as above before the addition of G-XDH. After incubation with G-XDH, the actual number of G-XDH was determined. As a typical example, the actual number of ZS-XR and G-XDH in the cavity of DNA scaffold (I-4XR/II-4XDH) in Figure 2 was estimated to be 3.8 molecules and 4.7 molecules, respectively. [The actual numbers of ZS-XR] = = 3.8 [The actual numbers of G-XDH] = ( ) = 4.7 S13

14 Figure S6. Determination of the actual number of ZS-XR molecules on the DNA scaffold. (a) (top) Illustration of the DNA scaffold (4-2-1-XR) containing four binding sites with BG modification for ZS- XR in cavity I, two in cavity II, and one in cavity III. (bottom) An AFM image of ZS-XR bound to the DNA scaffold XR. (b) Frequency distributions of molecular volumes of ZS-XR (top) for cavity III with one binding site for ZS-XR, (middle) for cavity II with two, and (bottom) for cavity I with four were shown, respectively (n = 100). (c) Standard curve for the number of binding sites for ZS-XR, which indicated the maximum number of bound ZS-XR molecules, versus molecular volumes. [4-2-1-XR] = 13.6 nm, [ZS-XR] = 300 nm, Reactions were carried out for 30 min on ice. S14

15 Figure S7. Determination of the actual number of G-XDH molecules on the DNA scaffold. (a) (top) Illustration of the DNA scaffold (4-2-1-XDH) containing four binding sites for the G-XDH dimer in cavity I, two in cavity II, and one in cavity III. (bottom) An AFM image of G-XDH dimers bound to the DNA scaffold XDH. (b) Frequency distributions of molecular volumes of the G-XDH dimers (top) for cavity III with one binding site for the G-XDH dimer, (middle) for cavity II with two, and (bottom) for cavity I with four were shown, respectively (n = 100). (c) Standard curve for the number of binding sites, which indicate the maximum number of dimeric G-XDH molecules, versus main peak of the molecular volume. [4-2-1-XDH] = 13.6 nm, [G-XDH] = 300 nm. Reactions were carried out for 30 min on ice. S15

16 Figure S8. Estimating the actual number of ZS-XR molecules bound on I-4XR. (a) Illustration of the DNA scaffold (I-4XR) containing four binding sites for ZS-XR (red) inside cavity I. (b) An AFM image of ZS-XR bound to the DNA scaffold I-4XR. (c) Frequency distribution of molecular volumes of ZS-XR (n = 161) for cavity I of I-4XR. [The actual numbers of ZS-XR] = = 3.9 S16

17 Figure S9. Effect of the DNA scaffold on the catalytic activity of ZS-XR. (a) Illustrations of the DNA scaffolds without holding the binding site (NB; top) and holding four binding sites with the BG modification for ZS-XR inside cavity I (I-4XR; bottom). (b) Time-course profiles of the oxidation of NADH monitored by absorbance at 340 nm. Enzyme assay was conducted in a solution containing ZS- XR in the absence (open triangle) or presence of the DNA scaffold (open circle: I-4XR; plus: NB). A solution of the DNA scaffold (when present, 4 nm), ZS-XR (50 nm), and xylose (200 mm) was incubated on ice for 30 min in a buffer (ph 7.6) containing 40 mm Tis, 20 mm acetic acid, 12.5 mm MgCl 2, 100 mm NaCl, 1 µm ZnCl 2, and 0.02% Tween-20. The resulting solution was used without further purification by gel filtration. Reactions were started with an addition of NADH (0.15 mm) at ambient temperature. S17

18 Figure S10. Effective removal of the unbound ZS-XR upon purification of the ZS-XR bound DNA scaffold. (a) Illustrations of the DNA scaffolds without holding the binding site (NB; left) and holding four binding sites with the BG modification for ZS-XR inside the cavity I (I-4XR; right). (b) Time-course profiles of the oxidation of NADH monitored by absorbance at 340 nm. Enzyme assay was conducted in a S18

19 solution containing ZS-XR in the absence (open green triangle) or presence of the DNA scaffold (open red circle: I-4XR; blue plus: NB). A solution of the DNA scaffold (when present, 12 nm), ZS-XR (300 nm), -mecaptoethanol (5 mm), and ZnCl 2 (1 µm) was incubated on ice for 30 min. The resulting solutions were purified by gel filtration as described in Materials and Methods and the eluted fractions were used for enzyme assay. The enzyme assay was conducted in the presence of xylose (200 mm) and NADH (0.05 mm) with the DNA scaffold assembled with ZS-XR (2.6 nm). (c) The catalytic activities of ZS-XR with or without the DNA scaffold after purification by gel filtration. One unit is defined as the amount of enzyme that consumes 1 µmol NADH per minute. Error bars were generated as the standard deviation of the mean from at least three replicates. The catalytic activity of ZS-XR was only observed when the DNA scaffold with four binding sites for ZS-XR was applied. These results strongly indicated that the purification procedure by gel filtration removed the unbound ZS-XR from the DNA scaffold. S19

20 Figure S11. Illustrations of coassembled ZS-XR and G-XDH on the DNA scaffolds with various interenzyme distances. Based on the molecular models for the complex of ZS-XR or G-XDH with their target sequences (Figure S1) and the design of DNA scaffolds, interenzyme distances of the coassembled ZS-XR and G-XDH were deduced to be (a) 10 nm, (b) 54 nm, and (c) 98 nm, respectively. The red and blue circles indicated the plausible ranges of motions of ZS-XR and G-XDH, respectively. S20

21 Figure S12. Specific binding ability and the actual number of enzymes inside cavity I of the DNA scaffold (I-4XR/4XDH). (a) An illustration of ZS-XR and G-XDH coassembled inside cavity I of I- 4XR/4XDH (top). An AFM image indicates the coassembly of ZS-XR and G-XDH inside cavity I of I- 4XR/4XDH (bottom). (b) Populations of ZS-XR and the coassembly of ZS-XR and G-XDH inside cavity I of the DNA scaffold (I-4XR/4XDH) were estimated by counting the number of cavities occupied by enzymes in AFM images. Population of the nonspecifically bound enzymes in the other cavities was also shown. (c) Frequency distribution of the molecular volumes of enzymes bound inside cavity I of the DNA scaffold (n =418). The numbers of monomeric G-XDH are shown. (d) The actual numbers of ZS-XR and G-XDH monomer were estimated from frequency distributions of molecular volumes for each enzyme. To determine the number of each enzymes in the same cavity, the yield of occupied cavity and the volume of the assembled ZS-XR were determined prior to coassemble ZS-XR and G-XDH. The yield was determined to be 95% (Figure S12b) and the volume analysis data showed only the fraction identified as four molecules of ZS-XR as shown in Figure S8. Upon addition of G-XDH, samples were analyzed by S21

22 AFM, and the volume of enzymes in the cavity was obtained. The increased volume over the four ZS-XR molecules was considered as the volume of assembled G-XDH. Frequency distribution of the molecular volumes of enzymes bound inside the cavity (Figure S12c) was analyzed as described above. The yield of coassembled enzymes and the actual numbers of ZS-XR and G-XDH monomer were determined to be: [Coassembled yield] = ( ) = 75% [The actual numbers of ZS-XR] = = 3.8 [The actual numbers of G-XDH] = ( ) = 4.2 S22

23 Figure S13. Specific binding ability and the actual number of enzymes inside the cavities of the DNA scaffold (I-4XR/II-4XDH). (a) An illustration of ZS-XR and G-XDH coassembled on I-4XR/II- 4XDH (top). An AFM image indicates the coassembly of ZS-XR and G-XDH inside cavities I and II, respectively, of I-4XR/II-4XDH (bottom). (b) Populations of ZS-XR and the coassembly of ZS-XR and G-XDH bound to the target sites were estimated by counting the number of cavities I and II occupied by enzymes in the AFM images. Population of the nonspecifically bound enzymes to other regions of the DNA scaffold was also shown. (c d) Frequency distributions of the molecular volumes of (c) ZS-XR (n S23

24 =117) bound inside cavity I and (d) G-XDH (n = 117) bound inside cavity II of the DNA scaffold. The numbers of monomeric G-XDH are shown. (e) The actual numbers of ZS-XR and G-XDH monomer were estimated from frequency distributions of molecular volumes for each enzyme and determined by using equation 1 in the Note S2. [The actual numbers of ZS-XR] = = 4.0 [The actual numbers of G-XDH] = ( ) = 3.8 S24

25 Figure S14. Specific binding ability and the actual number of enzymes inside cavities I and III of the DNA scaffold (I-4XR/III-4XDH). (a) An illustration of ZS-XR and G-XDH coassembled on I- 4XR/III-4XDH (top). An AFM image indicates the coassembly of ZS-XR and G-XDH inside cavities I and III of I-4XR/III-4XDH, respectively (bottom). (b) Populations of ZS-XR and the coassembly of ZS- XR and G-XDH to the target sites were estimated by counting the number of cavities I and III occupied by ZS-XR and G-XDH, respectively, in the AFM images. Population for the nonspecifically bound enzymes to other regions of the DNA scaffold was also shown. (c d) Frequency distributions of the S25

26 molecular volumes of (c) ZS-XR (n =75) bound inside cavity I and (d) G-XDH (n = 75) bound inside cavity III of the DNA scaffold. The numbers of monomeric G-XDH are shown. (e) The actual numbers of ZS-XR and G-XDH monomer were estimated from frequency distributions of molecular volumes for each enzyme and determined by using equation 1 in the Note S2. [The actual numbers of ZS-XR] = = 4.0 [The actual numbers of G-XDH] = ( ) = 4.4 S26

27 Note S3. Calculation of the interenzyme distance between ZS-XR and G-XDH in the solution. The average distance separating molecules in the solution was calculated by equation 1. S1 Briefly, in a 1 M enzyme solution, there are molecules/liter (0.6 molecules/nm 3 ) or inverting, the volume per molecule is V = 1.66 nm 3 /molecule at 1 M. Therefore, for a concentration C, the volume per molecule is V = 1.66/C. If we take the cube root of the volume per molecule, the average distance (d) will indicate in the equation 2: 1.18/ eq. 2 where C is in molar and d is in nanometer. In a solution containing 21 nm of ZS-XR assembled on the DNA scaffold and 85 nm free dimeric G- XDH, the total concentration was 106 nm. Based on eq. 2, the average separation of molecules was ca. 249 nm. S27

28 Figure S15. Time-course profiles of the change of absorbance at 340 nm in the enzyme cascade reactions with various ZS-XR/G-XDH interenzyme distances for bimolecular diffusion. The sequential enzymatic reaction with various interenzyme distances (10 nm, 54 nm, 98 nm and 249 nm) were monitored by means of the time-dependent absorbance changes of NADH at 340 nm. As a control, a reaction of ZS-XR on the DNA scaffold without G-XDH was also monitored (ZS-XR only). The reactions were carried out in the presence of the ZS-XR assembled DNA scaffold (21 nm), G-XDH (85 nm), xylose (12.5 mm), and NADH (2.0 mm). Experimental conditions are shown in the Material and Methods. S28

29 Figure S16. Analyses of the metabolites formation by the enzyme cascade reactions for bimolecular diffusion by HPLC. (a d) The enzyme cascade reaction in the presence of [1-3 H]-xylose with the interenzyme distances of (a) 10 nm, (b) 54 nm, (c) 98 nm, and (d) 249 nm were analyzed by HPLC. In the HPLC chromatograms, peaks 1, 2, and 3 indicated xylulose, xylose, and xylitol, respectively. The reactions were carried out in the presence of the ZS-XR assembled DNA scaffold (21 nm), G-XDH (85 nm), xylose (12.5 mm), [1-3 H]-xylose (200 nm), and NADH (2.0 mm). Experimental conditions are shown in the Material and Methods. S29

30 Table S3. Quantitation of the metabolites by HPLC. Systems Metabolites (µm) Xylose Xylitol Xylulose ZS-XR ± ± ZS-XR/G-XDH (10 nm) ± ± ± 19 ZS-XR/G-XDH (54 nm) ± ± ± 29 ZS-XR/G-XDH (98 nm) ± ± ± 20 ZS-XR/G-XDH (249 nm) ± ± ± 6 S30

31 Figure S17. Distance dependency of the enzyme cascade reactions on the DNA scaffold for unimolecular (NAD + ) diffusion. (a) Illustration of the enzyme cascade reactions by ZS-XR and G-XDH coassembled on the DNA scaffold I-4XR/4XDH with unimolecular diffusion. (b) Time-course profiles of the enzyme cascade reactions were monitored by the absorbance at 340 nm when the enzymes were coassembled with interenzyme distances of 10, 54, and 98 nm, and for the free diffusion system, in which ZS-XR was located on the DNA scaffold while the second enzyme G-XDH was present in free solution (theoretically estimated interenzyme distance of 249 nm). (c)the time-course profiles of the amount of NADH regenerated by G-XDH. The reactions were carried out in the presence of ZS-XR assembled on the DNA scaffold (21 nm), G-XDH dimer (85 nm), xylose (12.5 mm), xylitol (300 mm), and NADH (2 mm). Experimental conditions are shown in the Materials and Methods. S31

32 Note S4. Normalization of the initial rate of NADH regeneration (V ini) by G-XDH and the amount of products on the DNA scaffold. When both the enzymes were coassembled in a distance dependent manner, the coassembly yield on DNA scaffold varied with the interenzyme distance (Figures S12d, S13e, and S14e); this may reflect the yield of NADH regeneration. To avoid any discrepancies in the cascade reactions due to the yield of coassembly, the amount of regenerated NADH and the V ini were normalized according to the equation 3: Where, Y obs is the observed V ini or the amount of products (regenerated NADH or the final product xylulose) that consists of contributions from both coassembled (Y coassembled) and unassembled (Y unassembled) enzymes ZS-XR and G-XDH. The P coassembled and P unassembled are the coassembled and unassembled fractions of ZS-XR and G-XDH on the DNA scaffolds, respectively (determined by volume analysis of AFM images as described in Figures S12 S14). The free diffusion reaction was used to determine Y unassembled in the coassembled system for the normalization. The determined Y coassembled was used as the normalized V ini or the amount of products (regenerated NADH or the final product xylulose) as listed in Tables S4 and S5. S32

33 Table S4.Reaction parameters for the enzyme cascade in bimolecular transport system. System Bimolecular transport (xylitol & NAD + ) Reaction parameters for the enzyme cascade V ini (µm min -1 ) Regenerated NADH (µm) Distance dependency 10 nm 54 nm 98 nm 249 nm Raw 1.1 ± ± ± ± 0.2 Normalized 1.5 ± ± ± ± 0.2 Raw 172 ± ± ± ± 4 Normalized 211 ± ± ± ± 4 Turnover frequency (min -1 ) 2.7 ± ± ± ± 0.02 Raw 246 ± ± ± ± 6 Xylulose (µm) Normalized 317 ± ± ± ± 6 Turnover frequency (min -1 ) 3.7 ± ± ± ± 0.04 Table S5. Reaction parameters for the enzyme cascade in unimolecular transport system. System Unimolecular transport (NAD + ) Reaction parameters for the enzyme cascade V ini (µm min -1 ) Regenerated NADH (µm) Distance dependency 10 nm 54 nm 98 nm 249 nm Raw 0.39 ± ± ± ± 0.09 Normalized 0.44 ± ± ± ± 0.09 Raw 189 ± ± ± ± 34 Normalized 237 ± ± ± ± 34 Turnover frequency (min -1 ) 3.0 ± ± ± ± 0.5 S33

34 Note S5. Xylitol or NAD + diffusion model between ZS-XR and G-XDH. Three dimensional diffusion of intermediate, xylitol or NAD +, between enzymes was described by using three-dimensional Brownian motion to simulate distance dependency. S2 The equation 4 describes the convolution function of three dimensional Brownian motion of xylitol or NAD + with a constant catalytic rate for ZS-XR in the given time t S3. Thus, the equation 5 indicates the total amount of the available xylitol or NAD + to get around to G-XDH within the given time t. where n(r, t) is the concentration of intermediates (xylitol or NAD + ) at distance r from the initial position (ZS-XR), S(r, t) is the total amount of available intermediates (xylitol or NAD + ) to get around to G-XDH at the distance r from the initial position, D is the diffusion coefficient, and t is the diffusion time. ZS-XR reduces xylose and oxidizes the cofactor NADH to yield xylitol and NAD + at constant rate, k cat. τ is the average time between ZS-XR turnovers (1/k cat). S34

35 Figure S18. Model of xylitol or NAD + diffusion in a single ZS-XR/G-XDH pair. (a) Simplified illustration of the distant dependency (r) of xylitol or NAD + concentration gradient resulting from the three dimensional Brownian diffusion. (b) Total amounts of the available intermediate [xylitol (red) or NAD + (black)] to get around to G-XDH at the distance r from ZS-XR as a function of the interenzyme distance from 0 to 500 nm were calculated by using eq. 5 with the following parameters: Diffusion coefficient: S4,S5 + D NAD = µm 2 s -1 for NAD + ; D xylitol = µm 2 s -1, k cat (XR for xylose) = 305 s -1 ; τ = 1 / k cat = ; and the integration time 1sec. The value of k cat for xylose (305 s - 1 ), which was lower than the one for NAD + (893 s -1 ), was used in this simulation. S35

36 Note S6. Concentration dependency for the efficiency of cascade reaction by coassembled enzymes on the DNA scaffold. To identify the concentration dependency for the efficiency of cascade reaction, the cascade reactions of the unimolecular (NAD + ) transport system were carried out with different concentrations of ZS-XR and G-XDH coassembled on the DNA scaffold (I-4XR/4XDH). The free diffusion condition at each concentration was also carried out to compare its reaction efficiency with that on the scaffold (Figures S19a-d). Normalized initial rates of NADH regeneration (V ini) at different concentrations of ZS-XR and G-XDH coassembled on DNA scaffold (I-4XR/4XDH) are shown in Table S6. The ratios for V ini by G- XDH on the scaffold to that in the free diffusion condition at each concentration was compared to evaluate the efficiency of cascade reaction (Figure S19e). The reaction was started with an addition of NADH (50 M) to a mixture of ZS-XR and/or G-XDH located on the DNA scaffold (final conc. of the DNA scaffold was set to 2.8, 5.5, 11, and 22 nm, respectively), xylose (200 mm), and xylitol (300 mm) in a buffer (ph 7.0) containing 100 mm NaCl, 1 M ZnCl 2 and 0.02% Tween-20. The progress of reactions was monitored by measuring the time-course changes of absorbance at 340 nm. S36

37 Figure S19. Comparison of the reaction efficiency of ZS-XR/G-XDH on the DNA scaffold and in bulk solution at various concentrations of enzymes. Time-dependent changes in the absorbance at 340 S37

38 nm (a-d, left) and the amount of NADH regenerated by G-XDH (a-d, right) were analyzed for various concentrations of ZS-XR and G-XDH coassembled on the DNA scaffold (I-4XR/4XDH) or in bulk solution. The interenzyme distance on the DNA scaffold was fixed to 10 nm. The concentrations of coassembled ZS-XR/G-XDH on I-4XR/4XDH or in the bulk solution were set to (a) 2.8, (b) 5.5, (c) 11, and (d) 22 nm, respectively. (e) The ratio of normalized V ini for ZS-XR and G-XDH on the DNA scaffold (I-4XR/4XDH) over V ini for freely diffused ZS-XR and G-XDH in the bulk solution was evaluated at each enzyme concentration. Efficiency of the cascade reaction for coassembled ZS-XR and G-XDH on the DNA scaffold I- 4XR/4XDH with 10 nm interenzyme distance was compared to that for freely diffused enzymes. The average interenzyme distance in the bulk solution depended on the concentrations of enzymes as described in Note S3. On the other hand, the interenzyme distance of coassembled ZS-XR and G-XDH on I-4XR/4XDH was independent of the enzyme concentration at high dilution conditions. The ratios of V ini on the scaffold to that in the bulk solution were 8.5 (2.8 nm), 8.3 (5.5 nm), 6.1 (11 nm) and 3.6 (22 nm), respectively. The results indicated that the cascade reaction by coassembled ZS-XR and G-XDH on I- 4XR/4XDH proceeded more efficiently than that by freely diffused ZS-XR and G-XDH at low enzyme concentrations. By increasing the concentrations of enzymes, efficiency of the cascade reaction by freely diffused enzymes would approach to that of the coassembled ZS-XR and G-XDH on I-4XR/4XDH. Note: Though the efficiency of the sequential reaction on the DNA scaffold increased with lowering the concentration of enzyme, the reaction conditions in Figures 4 and 5 were selected to ensure accurate measurements of the final product xylulose by HPLC analysis in the bimolecular substrates diffusion system. S38

39 Table S6. Reaction parameters for the enzyme cascade with varying concentrations of enzymes/dna scaffold in the unimolecular transport system. System In bulk solution On the DNA scaffold (interenzyme distance : 10 nm) Parameters for activity of enzyme cascade Concentration of G-XDH and ZS-XR on the DNA scaffold (I- 4XR/4XDH) Actual [ZS-XR] on the DNA scaffold (nm)* [monomeric G-XDH] (nm) Calculated interenzyme distance (nm) V ini (3.0 ± 0.6) 10-2 (3.5 ± 0.7) 10-2 (10.5 ± 0.9) 10-2 (37.6 ± 13.0) 10-2 (µm/min) Raw Actual [ZS-XR] on the DNA scaffold (nm) * Actual [monomeric G- XDH] on the DNA scaffold (nm) ** V ini (µm/min) Raw Normalized (22.9 ± 3.1) 10-2 (22.4 ± 1.4) 10-2 (54.6 ± 8.1) 10-2 (29.6 ± 3.9) (28.8 ± 1.6) (69.3 ± 10.5) (126.9 ± 31.7) 10-2 (156.7 ± 37.9) 10-2 * [ZS-XR] on the DNA scaffold = (actual number of ZS-XR on the DNA scaffold) [DNA scaffold] ** [monomeric G-XDH] on the DNA scaffold = (actual number of monomeric G-XDH on the DNA scaffold) [DNA scaffold] Note: Actual number of ZS-XR and monomeric G-XDH on the DNA scaffold (I-4XR/4XDH) were shown in Figure S12. S39

40 Table S7. Michaelis constants of ZS-XR. Parameters Kinetic parameters of ZS-XR Xylose (a) NADH (b) K m (M) (145 ± 53) 10-4 (176 ± 19) 10-6 k cat (s -1 ) 305 ± ± 85 k cat/k m (M -1 s -1 ) (2.3 ± 1.2) 10 4 (5.1 ± 0.07) 10 6 V max (M s -1 ) (7.6 ± 1) 10-6 (2.2 ± 0.2) 10-5 (a) Concentrations of xylose were varied from 0 to 200 mm; and that of NADH was kept at 300 µm for the assay. (b) Concentrations of NADH were varied from 0 to 300 µm, and that of xylose was kept at 200 mm for the assay. The enzyme assays were carried out at the enzyme concentration of 25 nm in a buffer (ph 7.0) containing 40 mm Tris-HCl, 20 mm acetic acid, 12.5 mm MgCl 2, 100 mm NaCl, 1 µm ZnCl 2 and 0.02% Tween-20. Table S8. Michaelis constants of G-XDH. Kinetic parameters of G-XDH Parameters Xylitol (a) NAD + (b) K m (M) (906 ± 80) 10-4 (74 ± 6.3) 10-6 k cat (s -1 ) 180 ± ± 6.7 k cat/k m (M -1 s -1 ) (1.7 ± 0.02) 10 3 (1.7 ± 0.2) 10 6 V max (M s -1 ) (4.5 ± 0.4) 10-6 (3.1 ± 0.2) 10-6 (a) Concentrations of xylitol were varied from 0 to 800 mm, and that of NAD + was kept at 1.8 mm for the assay. (b) Concentrations of NADH were varied from 0 to 1.8 mm, and that of xylitol was kept at 300 mm for the assay. The enzyme assays were carried out at the enzyme concentration of 25 nm in a buffer (ph 7.0) containing 40 mm Tris-HCl, 20 mm acetic acid, 12.5 mm MgCl 2, 100 mm NaCl, 1 µm ZnCl 2 and 0.02% Tween-20. S40

41 Table S9. Nucleotide sequences of primer pairs for construction of an expression vector for ZS-XR. primer from 5 to 3 1_F_pri_NdeI-zif 2_R_pri_zif(+ XR) 3_F_pri_XR(+zif) 4_R_pri_XR_His_BamHI 5_F_pri_KpnI-XR 6_R_pri_XR_HindIII AATAATAATCATATGATGTGCAAAACCGGGGAGAAAC CAGAGTTCAACTTAATAGAAGGCATAGAACCACCAGAACCACCAGAACCACCGAATTCCTT CTCACCGGTGTGGATTTTG CAAAATCCACACCGGTGAGAAGGAATTCGGTGGTTCTGGTGGTTCTGGTGGTTCTATGCCT TCTATTAAGTTGAACTCTG ATATATGGATCCTCAATGATGATGATGATGATGGCCGCCGACGAAGATAGGAAT TAATTAGGTACCGGTGGTTCTGGTGGTTCTGGTGGTTC TAAAATAAGCTTTCAATGATGATGATGATGATGGCCGCCGAC S41

42 Table S10. Unmodified staple strands used for the assembly of the DNA origami scaffolds. The staple strands containing the binding sites for ZS-XR and G-XDH are highlighted in red. Oligo DNA Sequence (from 5 to 3 ) 001 GCGGGAGGTTTTGAAGCGAGGCGTTTTAGCGACATGTTCA 002 GCTAATGCATTCTGTCCAGACGACTCTTCTGACCTAAATTTTTTTCAA 003 ATATATTTAGATGATGAAACAAACTATTCATTTCAATTACCTTTTACA 004 TCGGGAGAACGTCAGATGAATATAGAAGGGTTAGAACCTAATTGTTTG 005 GATTATACAAAGAAACCACCAGAATTTGAGTAACATTATCATTTAGAA 006 GTATTAGAGAGCCGTCAATAGATAGTTGGCAAATCAACAGTATCAAAC 007 CCTCAATCAAAGGGACATTCTGGCAGATTCAC 008 CAGTCACAAACGGTACGCCAGAATGAGGCCGATTAAAGGG 009 GTAAAGTAAGAACGCGCCTGTTTCGGTATTCTAAGAACGCCTTAAA 010 AAACAAAAAAGAACGCGAGAAAACTAATGGTTTGAAATAGACAAAAG 011 CAGGTTTAAACAATAACGGATTCTCGCGCAGAGGCGAATATCAAGA 012 AATTATCACATCAATATAATCCTGCCATATCAAAATTATTAGATTTT 013 ATAGATTACTTTACAAACAATTCTTATTAATTTTAAAAGGGAGCGG 014 GATAGAACCTTGCTGAACCTCAAATTGAAAGGAATTGAGAACAACTA 015 CTAAACAGCCTGAGAAGTGTTTTGATTATTTACATTGGCCAACAGA 016 TCAAGATTAGTTGCTATTTTGCACTATAGAAGGCTTATCATCAACAA 017 TAGATAAGAGAATATAAAGTACCCCGACCGT 018 GTGATAAATCCAATCGCAAGACATTAATTAC 019 ATTTAACATACCAAGTTACAAAAGCCTGATT 020 GCTTTGAAAATAAAGAAATTGCGTTGCACGT 021 AAAACAGACAGATGATGGCAATTTCATATTC 022 CTGATTATTCCTTTGCCCGAACGGACAACTC 023 GTATTAAAATCTTTAGGAGCACTGAAGGTTA 024 TCTAAAATAATCTAAAGCATCACCCTTCTGA 025 CCTGAAAGCAATCGTCTGAAATGTATAATCA 026 GTGAGGCCCCTCGTTAGAATCAGAGCGGGAG 027 AAATCAGACCAGCTACAATTTTATCCTGAATC 028 TAAATAAGTCGAGCCAGTAATAAGTCCTGAACAAGAAAAATAGCAAGC 029 ATTTCATTTGAATTACTAAATGCTGATGCAAATAAGGCGT 030 TTGACGCTCGTAAGAATACGTGGCCCAGCAGCAAATGAAA 031 CGTGCTTTACCGAGTAAAAGAGTCCCTACATT 032 TTACCAACCATTACCGCGCCCAATAATATCC 033 CATCCTAATAGGCAGAGGCATTTAATAAACA 034 CCGGAATCTTATATAACTATATGCTTTTTTAA 035 GCTGAGAGACAGACAATATTTTTAACGCTCATGGAAATA 036 TGTCCATCACGCAAAGGTTGCTTTGACGAGCACGTATAA 037 GTAGGAATGCTAACGAGCGTCTTTCCAGAGC 038 ATGTAATTTTTACGAGCATGTAGTTTTCATC 039 TGGAAACAGTACATAAATCAATTCCGGCTTAGGTTGGGATAATTACTAGAAAAACGCCAAC 040 AGCTGCATTGCCGGAAACCAGGCACGGCGGA S42

43 041 CGCCGCTACAGGGCGCGTACTATTTAACCGT 042 CTAATTTGAAGCAAGCCGTTTTTAAAACCAAT 043 CAATAATCATCGCCATATTTAACAAGCCTGTT 044 TAGTATCAGACTACCTTTTTAACCATATGTGAGTGAATAA 045 TGAGGCGGTCAGTATTTTAATGCGCGAACTGACAATATTA 046 CCGCCAGCTACTTCTTTGATTAGTGCTTAATG 047 CGAGAACCCAGTTACAAAATAAACAGCCAT 048 ATTGAGAGGCTGTCTTTCCTTACGCACTCAT 049 TCTGAGATATGCGTTATACAAATAGGGCTTA 050 TTTAGTTCCTTGCTTCTGTAAAAATCATAGG 051 CTACTAATGACCATTAGATACAACGAGTAGA 052 AATCATATAGTAGTAGCATTAAGCATCAATT 053 TAATACTCAGGCAAGGCAAAGACATCCAATA 054 ACGCAAGTTTGCGGGAGAAGCCATGACCCTG 055 ACAGAGGGATAAAAATTTTTAGTTTATTTCA 056 TCCAGAATAGCCCTAAAACATCGAAGATAAA 057 CCGCCGCAATAACATCACTTGCCTGGTAATA 058 ATTATTTATATTAAACCAAGTACTCATTCCA 059 AGAACGGGGCCAACGCTCAACAGTTCTTACC 060 AGTATAAATAGTGAATTTATCAATCGTCGCT 061 ATTAATTATTCCCAATTCTGCGATTTCGCAA 062 ATGGTCAACGAGCTGAAAAGGTGTTTGCGGA 063 TGGCTTAGTGATAAGAGGTCATTATTAGCAA 064 AATTAAGCTGTACCAAAAACATTGAAGCCCG 065 AAAGACTTCAAAAAGATTAAGAGAACCCTCA 066 TATATTTTAACGAACCACCAGCAGCCATTAA 067 AAATACCGAAACTATCGGCCTTGCTGAGTAG 068 AAGAACTCTCACGCTGCGCGTAACCACCACAC 069 TTGAGTAAAAGGTGAATTATCACCCGGAAATTATTCATTATCCCAATC 070 TTAGAATCCGCTGAGAAGAGTCAACAGTGCCCGTATAAACTCAGTGCC 071 TTGGGGCGTAACCTGTTTAGCTATTCCATATAACAGTTGAATTTTCCC 072 CTCAGAGCCTTTAATTGCTCCTTTAGCTTAATTGCTGAATATTTTCAT 073 GATTGCATCAAATATCGCGTTTTAATAAAGCTAAATCGGTAATAAAGC 074 CAGTATCGTCTGCCAGTTTGAGGGAAATGCAATGCCTGAGGCAAAGCG 075 GTGTAGCGGTTTCACCAGTGAGACGCCAGGGTGGTTTTTCTGACGACGA 076 CAAATAAGAAACGATTTTTTGTTTGAAGGTAAATATTGAGTCACCGA 077 CTTGAGCCTAAGTTTTAACGGGGAGTTAATG 078 CCCCCTGCTAGCTTAGATTAAGACTTGAAAA 079 CATAGCGAGTCTGGAAGTTTCATGCAACTAA 080 AGTACGGTACATGTTTTAAATATATAATGCT 081 GTAGCTCAAGGATTAGAGAGTACCAAACTCC 082 AACAGGTCGAACCAGACCGGAAGATTCGAGC 083 TTCAAAGCCTATTATAGTCAGAATAATGTGT 084 AGGTAAAGCATCGTAACCGTGCAGCCTCAGG 085 AAGATCGCTTTGCGTATTGGGCGGGCAACAG S43

44 086 CTGATTGCGCGGGCGCTAGGGCGCTGGCAA 087 ATTAGAGAACCGATTGAGGGAGGAACGTCAAAAATGAA 088 CTATTTCGGAACCTAAGGAGTGTACTGGTAAATTTGGGA 089 GATGGGCGATTCAAAAGGGTGAGCAGGTCTTTACCCTGA 090 AGAGGCGGACTCCAGCCAGCTTTTTGGTGTA 091 CGAAAGGACCTTCACCGCCTGGCGCGCGGGG 092 AATAGCAGCCTTTACACAAAAGGGCGACATTCCCAGCAAA 093 ATCACCAGTGGCTTTTGATGATACTTATTCTGAAACATGA 094 CGAGAATGACCATAAATCAAAAATAAAGGCCGGAGACAGTAATGGGAT 095 AGGTCACGCCGGCACCGCTTCTGGTAATGAATCGGCCAACCCTGAGAG 096 AGTTGCAGTGGCGAGAAAGGAAGGGAAGAAAG 097 TTACCATTTACCAGCGCCAAAGAGAGAGAATAACATAAA 098 AAGTATTAAGAGGCTGAGTAAGCGTCATACATAGCACCA 099 TTGACCGTCAAATCACCATCAATTCAGAAAA 100 CAATTCCAGTTGGGTAACGCCAGATCAGCTC 101 GGCGAACGCAAGCGGTCCACGCTGTCGTGCC 102 AACAGGGAAGCGCATGAAAATTCATATGGTTAGCAAGGC 103 CGGAAACCTGAATTTACCGTTCCAGACTCCTCAAGAGA 104 AATGCTTTAAACAGTATGATATTCAACCGTTTCTCCGTGGGAACAAAAAGCGCC 105 ATTCGCCACCAGTCGGGAAACCTGGTTTGCC 106 CCAGCAGGTTTAGAGCTTGACGGGGAAAGCC 107 TGAAACCATTTGTCACAATCAATATAGACGGG 108 GATTAGCGATGGAAAGCGCAGTCTGTCACCAA 109 GGTTTAGTATAGGTGTATCACCGTAGGATTAG 110 CTCAGAACACCGCCACCCTCAGAAACTCAGGA 111 TACAAACTCGTAACACTGAGTTTCCCGCCACC 112 CAGCCCTCACAACGCCTGTAGCATGTCACCAG 113 AACAGTTTATGGGATTTTGCTAAATCCACAGA 114 TAAATTAACATTGAATCCCCCTCACAACTTTC 115 GCGCAACTTAACAACCCGTCGGATCTAGCTGA 116 GCCCCCGACGAAAATCCTGTTTGAGCTCACTGCCCGCTTTTTCAGGCT 117 AGAATTAACTGAACACCCTGAACCACGGAA 118 TAAGTTTATTCGATAGCAGCACCGCCTCATT 119 AAAGCCAGAGGGTTTTGCTCAGTAATAAGTA 120 TAGCCCGGAGATCGTCACCCTCAGAAGGCCG 121 CTTTTGCGGCGCCACCCTCAGAGCATAGGAA 122 CCCATGTACCGAGGTGAATTTCTTTTTATCA 123 GCTTGCTTTATAGTTAGCGTAACGTAAATGA 124 ATTTTCTGTCAGCGGAGTGAGAATAATCGTC 125 ATAAATATTTGCCGGAGAGGGTAGATTAAAT 126 GTGAGCGAGGTTGGGAAGGGCGATACATTAA 127 TTGCGTTGCTGGTGGTTCCGAAATCACTAAATCGGAACCCTAAAGGGA 128 AAAGACACAAAGTCAGAGGGTAATTGAGCGCT 129 AAATAAATTAATCAGTAGCGACAGAGAAACGC 130 GGGTTGATCCAGGCGGATAAGTGCTCACAAAC S44

45 131 GGGAGTTACAGCGAAAGACAGCATCGTCGAGA 132 CAAGCCCACACCACCCTCATTTTCGGCTTGCA 133 TGTATCGGAAACAGCTTGATACCGAGGGATAG 134 GACGTTAGATCTAAAGTTTTGTCGCCTTTAAT 135 TACTGCGGAGAAAGGAACAACTAATCTTTCCA 136 TCATCAACCTATTTTTGAGAGATCGCGTCCAA 137 GCTAACTCCGGTGCGGGCCTCTTCGCCAGCTT 138 CGTAAAGCGGCAAAATCCCTTATATGAGTGA 139 AATATCAGGTGGCAACATATAAAAATCAAGT 140 TTGCCTTTGATTGGCCTTGATATTTGAGGAC 141 TAAAGACTACGGCTACAGAGGCTCGGAACGA 142 GGGTAGCAATATTCGGTCGCTGACCCACGCA 143 TAACCGATTGACAACAACCATCGATAGTTGC 144 GCCGACAAAGGCTCCAAAAGGAGAAAATCTC 145 CAAAAAAAAATTTTTTCACGTTGAGGAATTG 146 CGAATAATTGTTTAGACTGGATATACAAAGG 147 CTATCAGGTCTGGCCTTCCTGTAGCTATTAC 148 GCCAGCTGAGCCTGGGGTGCCTAAAATCAAA 149 AGAATAGCTCAAGTTTTTTGGGGTCGAGGTGC 150 ACTGTAGAAATACATACATAAAGAGAGATAA 151 TTTTCATGAGGAAGTTTGAGGCAGGTCAGACAGCGTCAG 152 AATTCGCGTCATTGCCTGAGAGTGGGGGTAATAGTAAAA 153 AAGTGTAAGCGAAAGGGGGATGTTCAAAAAT 154 CACCCAAACCGAGATAGGGTTGAGAAGCATA 155 CCCACAAGAATTGAGTTAAGCCCTGTTAGCA 156 AACGTAGACGCGTTTTCATCGGCACAGCATTGACAGGAGGTTCCATTA 157 TTTTGCAAAAGAAGTTTTGCCAGACTGGAGCAAACAAGAGACCAATAGGAACGCCAGCTGCAAG 158 GCGATTAACACAACATACGAGCCGGTGTTGTT 159 CCAGTTTGGCGATGGCCCACTACGTGAACCAT 160 CATAGCCCCCTTATTACGCAGTAAATAATAAGAGCAAGAAACAATGA 161 AACGGGTAAAATACGTAATGCCACCACCAGAGCCGCCGCTTTTCGGT 162 ATTTTTTAAATCGATGAACGGTACGAGAGGC 163 TGTAGCAACATTGCAACAGGAAAGAATGGCTATTAGTCTAACACCGCCTGCAACAGTGCCAC 164 CTATCAGGGAACAAGAGTCCACTTCCGCTCA 165 AATAGCAATGGCATGATTAAGACTCCTTATTA 166 GCGTTTGCCCGCCACCAGAACCACTACGAAGGCACCAACC 167 TAAAAACCAAAATAGATCGTAAAACTAGCATTTAAATTTTTGTTAAGGTTTTCC 168 CAGTCACGTGTGTGAAATTGTTAATTAAAGA 169 ACGTGGACAACGTCAAAGGGCGAAAAACCGT 170 AAAAGAACTAGCTATCTTACCGAAGCCCTTTT 171 CCTCAGAGCATCTTTTCATAATCAGAATACCC 172 CGCCTGATTAAAACGAAAGAGGCAGCCACCAC 173 GGACAGATAAATTGTGTCGAAATCTGTATCAT 174 TGCTCATTGAACGGTGTACAGACCTTGAAAGA 175 TGCGATTTCAGTGAATAAGGCTTGAACAAAGC S45

46 176 ATCAGTTGTAAGAACTGGCTCATTTTACCTTA 177 GACGACGAAGATTTAGGAATACCAAAAGATTC 178 AATTCGCAGTCAATCATATGTACCGTTTACCA 179 CTGTTTCCACGTTGTAAAACGACGTTTGTTAA 180 TAAGAAAAAACGCAATAATAACGAAATCACC 181 GGAACCAGACCGCCACCCTCAGAAAAGAATA 182 CACTAAAAAGTACAACGGAGATTCGCGACCT 183 GCTCCATGCCGAACTGACCAACTAGGCGCAT 184 AGGCTGGCTACCCAAATCAACGTCCCTGACG 185 AGAAACACTTTAATCATTGTGAAATACCAGT 186 CAGGACGTCATTATTACAGGTAGCATTCAAC 187 TAATGCAGACTATCATAACCCTCCCGGTTGA 188 TAATCAGATGTAAACGTTAATATGCCAGTGC 189 CAAGCTTGCGAATTCGTAATCATGGTCATAG 190 ACCGAGGAGTAAGCAGATAGCCG 191 CCCTCAGAAGCCACCACCGGAACAGAAGGAA 192 CGAAACAACACTCATCTTTGACCAGCCGCCA 193 TAAGGGAATTACTTAGCCGGAACACCAAGCG 194 ATATTCATTGACCTTCATCAAGAGTCAATCA 195 ATTTCAACCAGAACGAGTAGTAAAGAACCGG 196 CGGAACAATGGGAAGAAAAATCTATGGTTTA 197 AGAGCAACATACATAACGCCAAACGAACTAA 198 ATTTAAATAAAGCCCCAAAAACAGCATAGTA 199 CCCCGGGTACCGAGCTCATGCCTGCAGGTCGAAGCAAAT Table S11. Modified nucleotide sequences for the staple strands containing the binding sites for G-XDH used for preparation of DNA scaffolds. Oligo DNA Sequence (from 5 to 3 ) 051- G-XDH CTACTAACTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGTGACCATTAGATACAACGAGTAGA 052- G-XDH AATCATACTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGTAGTAGTAGCATTAAGCATCAATT 053- G-XDH TAATACTCTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGCAGGCAAGGCAAAGACATCCAATA 054- G-XDH ACGCAAGCTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGTTTGCGGGAGAAGCCATGACCCTG 110- G-XDH CTCAGAACACCGCCACCCTCAGAACTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGACTCAGGA 111- G-XDH TACAAACTCGTAACACTGAGTTTCCTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGCCGCCACC 112- G-XDH CAGCCCTCACAACGCCTGTAGCATCTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGGTCACCAG 113- G-XDH AACAGTTTATGGGATTTTGCTAAACTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGTCCACAGA 173- G-XDH GGACAGATCTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGAAATTGTGTCGAAATCTGTATCAT 174- G-XDH TGCTCATTCTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGGAACGGTGTACAGACCTTGAAAGA 175- G-XDH TGCGATTTCTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGCAGTGAATAAGGCTTGAACAAAGC 176- G-XDH ATCAGTTGCTTCATGAGTCATGCGTTTTCGCATGACTCATGAAGTAAGAACTGGCTCATTTTACCTTA S46

47 Table S12. Modified nucleotide sequences for the staple strands containing the binding sites for ZS-XR with BG modification used for preparation of DNA scaffolds. Oligo DNA Sequence (from 5 to 3 ) 020-ZS-XR GCTTTGAACTTACGCCCACGCGCGTT BG TTCGCGCGTGGGCGTAAGAATAAAGAAATTGCGTTGCACGT 021-ZS-XR AAAACAGACTTACGCCCACGCGCGTT BG TTCGCGCGTGGGCGTAAGCAGATGATGGCAATTTCATATTC 022-ZS-XR CTGATTATCTTACGCCCACGCGCGTT BG TTCGCGCGTGGGCGTAAGTCCTTTGCCCGAACGGACAACTC 023-ZS-XR GTATTAAACTTACGCCCACGCGCGTT BG TTCGCGCGTGGGCGTAAGATCTTTAGGAGCACTGAAGGTTA 079-ZS-XR CATAGCGACTTACGCCCACGCGCGTT BG TTCGCGCGTGGGCGTAAGGTCTGGAAGTTTCATGCAACTAA 082-ZS-XR AACAGGTCCTTACGCCCACGCGCGTT BG TTCGCGCGTGGGCGTAAGGAACCAGACCGGAAGATTCGAGC 142-ZS-XR GGGTAGCACTTACGCCCACGCGCGTT BG TTCGCGCGTGGGCGTAAGATATTCGGTCGCTGACCCACGCA BG modified amino-c6-t was denoted as T BG. S47

48 References: S1. Erickson, H. P. Biol. Proced. Online 2009, 15, S2. Pathria, R. K. Statistical Mechanics, 2nd ed.; Butterworth-Heinemann: Woburn, MA, 1996; pp S3. Fu, J.; Liu, M.; Liu, Y.; Woodbury, N. W.; Yan, H. J. Am. Chem. Soc. 2012, 134, S4. Damian, A.; Omanovic, S. J. Mol. Catal. A: Chem. 2006, 253, 222. S5. Lebeau, T.; Jouenne, T.; Junter, G. A. Enzyme Microb. Technol. 1998, 22, 434. S48