リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

リケラボ 全国の大学リポジトリにある学位論文・教授論文を一括検索するならリケラボ論文検索大学・研究所にある論文を検索できる

リケラボ 全国の大学リポジトリにある学位論文・教授論文を一括検索するならリケラボ論文検索大学・研究所にある論文を検索できる

大学・研究所にある論文を検索できる 「Enzyme cascade reactions on 3D DNA scaffold with dynamic shape transformation」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

コピーが完了しました

URLをコピーしました

論文の公開元へ論文の公開元へ
書き出し

Enzyme cascade reactions on 3D DNA scaffold with dynamic shape transformation

LIN, PENG 京都大学 DOI:10.14989/doctor.k23437

2021.07.26

概要

細胞内の代謝反応が効率的に進行するためには、代謝に関わる酵素群が効率良く逐次反応を触媒する必要がある。逐次反応に関わる酵素を近接させると逐次反応が高効率で進行する現象は報告されているが、細胞内で見られるような区画化された環境にある酵素による代謝反応の詳細は明らかでない。本論文は、DNAナノ構造体を用いて形状が変化する足場を構築し、3次元的に区画化された空間に配置したキシロース代謝酵素による代謝反応を解析した結果を論じたもので、6章からなっている。

第1章は序論であり、生体内代謝経路において発見された階層的な酵素組織体と細胞内区画の特徴、そして細胞区画内での酵素による物質生産・エネルギー利用について論じている。細胞内代謝経路の高い反応効率と選択性を支配する化学を詳細に解析するためには、細胞外で酵素が密接した環境を構築する必要があることを指摘するとともに、これまでの細胞外で代謝経路を構築した研究を概説している。なかでも酵素を特定の空間に配置する足場としてDNAナノ構造体に注目し、活性を保ったまま定量的に酵素を1分子ずつナノメートル精度で固定する方法、さらにはこれまでにDNAナノ構造体を用いて構築された代謝経路の特徴とそれらの問題点を論じている。さらに、その方法により酵素を特定空間に配置して、人工的に構築した区画内での代謝経路反応を解析するという本論文の目的を述べている。

第2章では、二つの四角錐台が繋がった舟形DNAナノ構造体上の特定位置に、至適pHが異なるキシロース還元酵素(XR)とキシリトール脱水酵素(XDH)をモジュール型アダプターと融合することによって個別に配置した。両酵素ともにナノ構造体上に配置することによって反応速度が4倍に上昇した。これまでに、DNAナノ構造体に配置した酵素の反応が加速される理由として、①負に帯電した足場への基質吸着、②足場付近の局所pH変化による酵素活性の向上、③足場もしくは足場近傍の高密度水層による酵素安定化が提唱されている。本反応では基質および補酵素は中性もしくは負に帯電しているため①の仮説は成立しない。蛍光性pH指示薬を用いたナノ構造体上の局所pHを測定すると、XRとXDHの至適pHがそれぞれ6.0、8.0であることから、②の仮説にあるDNAナノ構造体表面に由来する局所pH変化は、ナノ構造体に配置された両酵素の反応速度増大の原因ではないことを明らかにした。さらに③の仮説を発展させて、酵素周辺で親水性基質の濃度が上昇する機構を提唱した。

第3章では、第2章で構築した舟形DNAナノ構造体を、四角錘台間での二本鎖DNA形成により六角柱構造体へと変換する方法を開発した。それぞれの四角錐台に導入した蛍光分子間の蛍光共鳴エネルギー移動を利用して、両構造体の存在比を求めるとともに、構造変換を実時間で観測した。XDHを配置した舟形DNAナノ構造体を、室温で12時間反応後、90%以上の収率で六角柱構造体へと変換し、六角柱構造体内部に配置した酵素の反応速度は、舟形構造体上の酵素と同等であることを明らかにした。

第4章では、第3章で開発した舟形DNAナノ構造体を六角柱構造体へと変換する方法を利用し、2種類の酵素XDHおよびキシルロースキナーゼ(XK)を配置した舟形DNAナノ構造体の構造変換によってXDHとXK間の酵素間距離を変化させた。これらの足場上で特定の酵素間距離にある2段階逐次反応と、それぞれ別の足場に配置したXDHおよびXKを用いた逐次反応の効率を評価したところ、20nmから1110nmの酵素間距離で第2段階の代謝回転数は10%しか減少しなかった。これは10nmから250nmへと酵素間距離を変化させると代謝回転数が90%以上減少するXRとXDHの代謝反応とは大きく異なっていた。これらの結果から、2段階目の反応に関与する酵素のミカエリス定数(Km)が1段階目より遙かに小さい場合、もしくは代謝回転数(kcat)が遙かに大きい場合には、第1段階と第2段階の反応に関与する酵素間距離は逐次反応速度に大きくは影響しないことを明らかにした。この結果は多段階の人工代謝経路を作製する上での重要な設計指針となる。

第5章では、より短時間で舟形DNAナノ構造体を六角柱構造体へと変換する方法を開発し、その方法を利用して熱的に不安定な酵素を含む人工代謝経路を作製した。舟形から六角柱へのDNAナノ構造体の構造変化は、短鎖DNAの鎖交換と二重鎖形成に依存している。短鎖DNAの鎖交換にtoehold機構を利用して、第3章の方法では12時間を要した構造変換を2時間以内に完了する方法を開発した。Kmとkcatの均衡がとれているXRとXDHの逐次反応を評価したところ、舟形構造体よりも六角柱構造体内部に配置した場合の方が代謝回転数は高かった。さらに、六角柱構造体内部に配置したXRとXDHの逐次反応は、平面状DNAナノ構造体に同じ酵素間距離で配置した場合よりも効率が高かった。これらの結果から、逐次代謝反応の効率は、Kmとkcatの均衡がとれている二つの酵素でも、酵素間距離だけでなく酵素の周辺環境にも依存すると結論づけた。

第6章は総括であり、細胞外で酵素を1分子ずつ3次元可動空間に配置して、細胞内を模した環境にある酵素の代謝反応を検証した結果を要約している。3次元空間に区画化された逐次反応に関与する酵素は、同じ酵素間距離で2次元的に配置された場合よりも、逐次反応速度が大きいことを明らかにし、その原因として区画化された酵素の周辺環境の水分子層の役割を指摘している。さらに、細胞内の酵素反応を理解する新しい知見として、そして細胞外で効率的に物質生産・エネルギー変換が可能な分子コンビナートの設計指針として、酵素間距離が逐次反応速度に影響を及ぼす上での酵素特性について要約している。

この論文で使われている画像

参考文献

Chapter1

1. Hossain, G. S. et al. Rewriting the metabolic blueprint: advances in pathway diversification in microorganisms. Front. Microbiol. 9, (2018).

2. Chen, A. H. & Silver, P. A. Designing biological compartmentalization. Trends Cell Biol. 22, 662–670 (2012).

3. Agapakis, C. M., Boyle, P. M. & Silver, P. A. Natural strategies for the spatial optimization of metabolism in synthetic biology. Nat. Chem. Biol. 8, 527–535 (2012).

4. Bonacci, W. et al. Modularity of a carbon-fixing protein organelle. Proc. Natl. Acad. Sci. 109, 478–483 (2012).

5. Savage, D. F., Afonso, B., Chen, A. H. & Silver, P. A. Spatially ordered dynamics of the bacterial carbon fixation machinery. Science 327, 1258–1261 (2010).

6. Wheeldon, I. et al. Substrate channelling as an approach to cascade reactions. Nat. Chem. 8, 299–309 (2016).

7. Schneider, T. R. et al. Loop closure and intersubunit communication in tryptophan synthase. Biochemistry 37, 5394–5406 (1998).

8. Dunn, M. F. Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex. Arch. Biochem. Biophys. 519, 154–166 (2012).

9. Miles, E. W., Rhee, S. & Davies, D. R. The molecular basis of substrate channeling. J. Biol. Chem. 274, 12193–12196 (1999).

10. Wu, F. & Minteer, S. Krebs cycle metabolon: structural evidence of substrate channeling revealed by cross-linking and mass spectrometry. Angew. Chem., Int. Ed. 54, 1851–1854 (2015).

11. Guynn, R. W., Gelberg, H. J. & Veech, R. L. Equilibrium constants of the malate dehydrogenase, citrate synthase, citrate lyase, and acetyl coenzyme A hydrolysis reactions under physiological conditions. J. Biol. Chem. 248, 6957–6965 (1973).

12. Berg, J. M., Tymoczko, J. L., & Stryer, L. Biochemistry. WH Freeman, New York, 2019.

13. Liu, L. N. Distribution and dynamics of electron transport complexes in cyanobacterial thylakoid membranes. Biochim. Biophys. Acta-Bioenerg. 1857, 256–265 (2016).

14. Šrejber, M. et al. Membrane-attached mammalian cytochromes P450: an overview of the membrane’s effects on structure, drug binding, and interactions with redox partners. J. Inorg. Biochem. 183, 117–136 (2018).

15. Laage, D., Elsaesser, T. & Hynes, J. T. Water dynamics in the hydration shells of biomolecules. Chem. Rev. 117, 10694–10725 (2017).

16. Klermund, L., Poschenrieder, S. T. & Castiglione, K. Biocatalysis in polymersomes: improving multienzyme cascades with incompatible reaction steps by compartmentalization. ACS Catal. 7, 3900–3904 (2017).

17. Walde, P. & Ichikawa, S. Enzymes inside lipid vesicles: preparation, reactivity and applications. Biomol. Eng. 18, 143–177 (2001).

18. Zhang, G., Quin, M. B. & Schmidt-Dannert, C. Self-assembling protein scaffold system for easy in vitro coimmobilization of biocatalytic cascade enzymes. ACS Catal. 8, 5611–5620 (2018).

19. Zhao, F., Li, H., Jiang, Y., Wang, X. & Mu, X. Co-immobilization of multienzyme on control-reduced graphene oxide by non-covalent bonds: an artificial biocatalytic system for the one-pot production of gluconic acid from starch. Green Chem. 16, 2558–2565 (2014).

20. Wei, Q., He, S., Qu, J. & Xia, J. Synthetic multienzyme complexes assembled on virus-like particles for cascade biosynthesis in cellulo. Bioconjug. Chem. 31, 2413–2420 (2020).

21. Hong, F., Zhang, F., Liu, Y. & Yan, H. DNA origami: scaffolds for creating higher order structures. Chem. Rev. 117, 12584-12640 (2017).

22. Tapio, K. & Bald, I. The potential of DNA origami to build multifunctional materials. Multifunct. Mater. 3, (2020).

23. Rajendran, A., Nakata, E., Nakano, S. & Morii, T. Nucleic-acid-templated enzyme cascades. ChemBioChem 18, 696–716 (2017).

24. Lim, S., Kim, J., Kim, Y., Xu, D. & Clark, D. S. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chem. Commun. 56, 4950– 4953 (2020).

25. Seeman, N. C. Nucleic acid junctions and lattices. J. Theor. Biol. 99, 237–247 (1982).

26. Seeman, N. C. & Sleiman, H. F. DNA nanotechnology. Nat. Rev. Mater. 3, (2017).

27. Yan, H., Park, S. H., Finkelstein, G., Reif, J. H. & LaBean, T. H. DNA-templated self-assembly of protein arrays and highly conductive nanowires. Science 301, 1882–1884 (2003).

28. Park, S. H. et al. Three-helix bundle DNA tiles self-assemble into 2D lattice or 1D templates for silver nanowires. Nano Lett. 5, 693–696 (2005).

29. Shih, W. M., Quispe, J. D. & Joyce, G. F. A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature 427, 618–621 (2004).

30. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

31. Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).

32. Douglas, S. M. et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001–5006 (2009).

33. Andersen, E. S. et al. Self-assembly of a nanoscale DNA box with a controllable lid. Nature 459, 73–76 (2009).

34. Han, D. et al. DNA origami with complex curvatures in three-dimensional space. Science 332, 342–346 (2011).

35. Dietz, H., Douglas, S. M. & Shih, W. M. Folding DNA into twisted and curved nanoscale shapes. Science 325, 725–730 (2009).

36. Han, D. et al. DNA gridiron nanostructures based on four-arm junctions. Science 339, 1412–1415 (2013).

37. Zadegan, R. M. et al. Construction of a 4 Zeptoliters switchable 3D DNA box origami. ACS Nano 6, 10050–10053 (2012).

38. Zhang, D. Y. & Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131, 17303–17314 (2009).

39. Zhang, D. Y. & Seelig, G. Dynamic DNA nanotechnology using stranddisplacement reactions. Nat. Chem. 3, 103–113 (2011).

40. Rangel, A. E., Hariri, A. A., Eisenstein, M. & Soh, H. T. Engineering aptamer switches for multifunctional stimulus-responsive nanosystems. Adv. Mater. (2020).

41. Turek, V. A. et al. Thermo-responsive actuation of a DNA origami flexor. Adv. Funct. Mater. 28, 1–7 (2018).

42. Kim, S. H. et al. Reversible regulation of enzyme activity by pH-responsive encapsulation in dna nanocages. ACS Nano 11, 9352–9359 (2017). 4

3. Marras, A. E. et al. Cation-activated avidity for rapid reconfiguration of dna nanodevices. ACS Nano 12, 9484–9494 (2018).

44. Kopperger, E. et al. A self-assembled nanoscale robotic arm controlled by electric field. Science 359, 296–301 (2018).

45. Deluca, M., Shi, Z., Castro, C. E. & Arya, G. Dynamic DNA nanotechnology: toward functional nanoscale devices. Nanoscale Horizons 5, 182–201 (2020).

46. Douglas, S. M., Bachelet, I. & Church, G. M. A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831–834 (2012).

47. Amir, Y., Ben-Ishay, E., Levner, D., Ittah, S., Abu-Horowitz, A., & Bachelet, I. Universal computing by DNA origami robots in a living animal. Nat. Nanotechnol. 9, 353-357 (2014).

48. Li, S. et al. A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivo. Nat. Biotechnol. 36, 258–264 (2018).

49. Grossi, G., Dalgaard Ebbesen Jepsen, M., Kjems, J. & Andersen, E. S. Control of enzyme reactions by a reconfigurable DNA nanovault. Nat. Commun. 8, (2017).

50. Juul, S. et al. Temperature-controlled encapsulation and release of an active enzyme in the cavity of a self-assembled DNA nanocage. ACS Nano 7, 9724– 9734 (2013).

51. Ijäs, H., Hakaste, I., Shen, B., Kostiainen, M. A. & Linko, V. Reconfigurable DNA origami nanocapsule for pH-controlled encapsulation and display of cargo. ACS Nano 13, 5959-5967 (2019).

52. Chandrasekaran, A. R., Anderson, N., Kizer, M., Halvorsen, K., & Wang, X. Beyond the fold: emerging biological applications of DNA origami. ChemBioChem 17, 1081-1089 (2016).

53 Yang, Y. R., Liu, Y. & Yan, H. DNA Nanostructures as programmable biomolecular scaffolds. Bioconjug. Chem. 26, 1381–1395 (2015).

54. Ngo, T. A. et al. Protein adaptors assemble functional proteins on DNA scaffolds. Chem. Commun. 55, 12428–12446 (2019).

55. Jaekel, A., Stegemann, P. & Saccà, B. Manipulating enzymes properties with DNA nanostructures. Molecules 24, (2019).

56. Kong, G. et al. DNA origami-based protein networks: from basic construction to emerging applications. Chem. Soc. Rev. 50, 1846–1873 (2021).

57. Mallik, L. et al. Electron microscopic visualization of protein assemblies on flattened DNA origami. ACS Nano 9, 7133–7141 (2015).

58. Shen, W., Zhong, H., Neff, D. & Norton, M. L. NTA directed protein nanopatterning on DNA origami nanoconstructs. J. Am. Chem. Soc. 131, 6660– 6661 (2009).

59. Yamazaki, T., Heddle, J. G., Kuzuya, A. & Komiyama, M. Orthogonal enzyme arrays on a DNA origami scaffold bearing size-tunable wells. Nanoscale 6, 9122–9126 (2014).

60. Chhabra, R. et al. Spatially addressable multiprotein nanoarrays templated by aptamer-tagged DNA nanoarchitectures. J. Am. Chem. Soc. 129, 10304–10305 (2007).

61. Valero, J., Pal, N., Dhakal, S., Walter, N. G. & Famulok, M. A bio-hybrid DNA rotor-stator nanoengine that moves along predefined tracks. Nat. Nanotechnol. 13, 496–503 (2018).

62. Ge, Z. et al. Constructing submonolayer DNA origami scaffold on gold electrode for wiring of redox enzymatic cascade pathways. ACS Appl. Mater. Interfaces 11, 13881–13887 (2019).

63. Zhao, S. et al. Efficient intracellular delivery of RNase a using DNA origami carriers. ACS Appl. Mater. Interfaces 11, 11112–11118 (2019).

64 Saccà, B. et al. Orthogonal protein decoration of DNA origami. Angew. Chem., Int. Ed. 122, 9568-9573 (2010).

65. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

66. Los, G. V. Halo-tag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373-382 (2008).

67. Pavletich, N. P. & Pabo, C. Zinc finger-DNA recognition : crystal structure of a zif268-DNA complex at 2.1 A. Science 252, 809–818 (1991).

68. Greisman, H. A. & Pabo, C. O. A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites. Science 275, 657–661 (1997).

69. Shaner, N. C., Steinbach, P. A., & Tsien, R. Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905-909 (2005).

70. Nakata, E. et al. Zinc-finger proteins for site-specific protein positioning on DNA-origami structures. Angew. Chem., Int. Ed. 51, 2421–2424 (2012).

71. Ellenberger, T. E., Brandl, C. J., Struhl, K. & Harrison, S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted α helices: crystal structure of the protein-DNA complex. Cell 71, 1223–1237 (1992).

72. Watanabe, S., Kodaki, T. & Makino, K. Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc. J. Biol. Chem. 280, 10340–10349 (2005).

73. Ngo, T. A., Nakata, E., Saimura, M., Kodaki, T. & Morii, T. A protein adaptor to locate a functional protein dimer on molecular switchboard. Methods 67, 142– 150 (2014).

74. Nakata, E., Dinh, H., Ngo, T. A., Saimura, M. & Morii, T. A modular zinc finger adaptor accelerates the covalent linkage of proteins at specific locations on DNA nanoscaffolds. Chem. Commun. 51, 1016–1019 (2015).

75. Ngo, T. A., Nakata, E., Saimura, M. & Morii, T. Spatially organized enzymes drive cofactor-coupled cascade reactions. J. Am. Chem. Soc. 138, 3012–3021 (2016).

76. Nguyen, T. M., Nakata, E., Saimura, M., Dinh, H. & Morii, T. Design of modular protein tags for orthogonal covalent bond formation at specific DNA sequences. J. Am. Chem. Soc. 139, 8487–8496 (2017). 7

7. Nguyen, T. M. et al. Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics. Chem. Sci. 10, 9315–9325 (2019).

78. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

79. Linko, V. et al. DNA-based enzyme reactors and systems. Nanomaterials 6, 139 (2016).

80. Wilner, O. I. et al. Enzyme cascades activated on topologically programmed DNA scaffolds. Nat. Nanotechnol. 4, 249–254 (2009).

81. Fu, J., Liu, M., Liu, Y., Woodbury, N. W. & Yan, H. Interenzyme substrate diffusion for an enzyme cascade organized on spatially addressable DNA nanostructures. J. Am. Chem. Soc. 134, 5516–5519 (2012).

82. Xin, L., Zhou, C., Yang, Z. & Liu, D. Regulation of an enzyme cascade reaction by a DNA machine. Small 9, 3088–3091 (2013).

83. Linko, V., Eerikäinen, M. & Kostiainen, M. A. A modular DNA origami-based enzyme cascade nanoreactor. Chem. Commun. 51, 5351–5354 (2015).

84. Zhao, Z. et al. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion. Nat. Commun. 7, (2016).

85. Fu, J. et al. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm. Nat. Nanotechnol. 9, 531–536 (2014).

86. Liu, M., Fu, J., Qi, X., Wootten, S., Woodbury, N. W., Liu, Y., & Yan, H. A three-enzyme pathway with an optimised geometric arrangement to facilitate substrate transfer. ChemBioChem, 17, 1097-1101 (2016).

87. Dinh, H. et al. Reaction of ribulose biphosphate carboxylase/oxygenase assembled on a DNA scaffold. Bioorganic Med. Chem. 27, 115120 (2019).

88. Frey, R., Mantri, S., Rocca, M. & Hilvert, D. Bottom-up construction of a primordial carboxysome mimic. J. Am. Chem. Soc. 138, 10072 (2016).

89. Zhang, Y., Ge, J. & Liu, Z. Enhanced activity of immobilized or chemically modified enzymes. ACS Catal. 5, 4503–4513 (2015).

90. Ellis, G. A. et al. Artificial multienzyme scaffolds: pursuing in vitro substrate channeling with an overview of current progress. ACS Catal. 9, 10812–10869 (2019).

91. Rudiuk, S., Venancio-Marques, A. & Baigl, D. Enhancement and modulation of enzymatic activity through higher-order structural changes of giant DNA-protein multibranch conjugates. Angew. Chem., Int. Ed. 124, 12866–12870 (2012).

92. Lin, J. L. & Wheeldon, I. Kinetic enhancements in DNA-enzyme nanostructures mimic the sabatier principle. ACS Catal. 3, 560–564 (2013).

93. Gao, Y. et al. Tuning enzyme kinetics through designed intermolecular interactions far from the active site. ACS Catal. 5, 2149–2153 (2015).

94. Timm, C. & Niemeyer, C. M. Assembly and purification of enzymefunctionalized DNA origami structures. Angew. Chem., Int. Ed. 54, 6745–6750 (2015).

95. Zhang, Y., Tsitkov, S. & Hess, H. Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade. Nat. Commun. 7, (2016).

96. Xiong, Y., Huang, J., Wang, S. T., Zafar, S. & Gang, O. Local environment affects the activity of enzymes on a 3D molecular scaffold. ACS Nano 14, 14646–14654 (2020).

Chapter2

1. Di Cosimo, R., Mc Auliffe, J., Poulose, A. J. & Bohlmann, G. Industrial use of immobilized enzymes. Chem. Soc. Rev. 42, 6437–6474 (2013).

2. Sheldon, R. A. Enzyme immobilization: the quest for optimum performance. Adv. Synth. Catal. 349, 1289–1307 (2007).

3. Zhang, Y., Ge, J. & Liu, Z. Enhanced activity of immobilized or chemically modified enzymes. ACS Catal. 5, 4503–4513 (2015).

4. Lim, S., Jung, G. A., Glover, D. J. & Clark, D. S. Enhanced enzyme activity through scaffolding on customizable self-assembling protein filaments. Small 15, 1–11 (2019).

5. Tabaei, S. R., Rabe, M., Zetterberg, H., Zhdanov, V. P. & Höök, F. Single lipid vesicle assay for characterizing single-enzyme kinetics of phospholipid hydrolysis in a complex biological fluid. J. Am. Chem. Soc. 135, 14151–14158 (2013).

6. Wang, Y. & Caruso, F. Mesoporous silica spheres as supports for enzyme immobilization and encapsulation. Chem. Mater. 17, 953–961 (2005).

7. Lin, P. et al. Assembly of graphene oxide-formate dehydrogenase composites by nickel-coordination with enhanced stability and reusability. Eng. Life Sci. 18, 326–333 (2018).

8. Virgen-Ortíz, J. J. et al. Polyethylenimine: a very useful ionic polymer in the design of immobilized enzyme biocatalysts. J. Mater. Chem. B 5, 7461–7490 (2017).

9. Rajendran, A., Nakata, E., Nakano, S. & Morii, T. Nucleic-acid-templated enzyme cascades. ChemBioChem 18, 696–716 (2017).

10. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

11. Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).

12. Grossi, G., Dalgaard Ebbesen Jepsen, M., Kjems, J. & Andersen, E. S. Control of enzyme reactions by a reconfigurable DNA nanovault. Nat. Commun. 8, (2017).

13. Wilner, O. I. et al. Enzyme cascades activated on topologically programmed DNA scaffolds. Nat. Nanotechnol. 4, 249–254 (2009).

14. Ngo, T. A. et al. Protein adaptors assemble functional proteins on DNA scaffolds. Chem. Commun. 55, 12428–12446 (2019).

15. Zhao, Z. et al. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion. Nat. Commun. 7, (2016).

16. Rudiuk, S., Venancio-Marques, A. & Baigl, D. Enhancement and modulation of enzymatic activity through higher-order structural changes of giant DNA-Protein multibranch conjugates. Angew. Chem., Int. Ed. 51, 12694–12698 (2012).

17. Timm, C. & Niemeyer, C. M. Assembly and purification of enzymefunctionalized DNA origami structures. Angew. Chem., Int. Ed. 54, 6745–6750 (2015).

18. Lin, J. L. & Wheeldon, I. Kinetic enhancements in DNA-enzyme nanostructures mimic the sabatier principle. ACS Catal. 3, 560–564 (2013).

19. Zhang, Y., Tsitkov, S. & Hess, H. Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade. Nat. Commun. 7, (2016).

20. Xiong, Y., Huang, J., Wang, S. T., Zafar, S. & Gang, O. Local environment affects the activity of enzymes on a 3D molecular scaffold. ACS Nano 14, 14646–14654 (2020).

21. Douglas, S. M., Bachelet, I. & Church, G. M. A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831–834 (2012).

22. Sander, B. & Golas, M. M. Visualization of bionanostructures using transmission electron microscopical techniques. Microsc. Res. Tech. 74, 642–663 (2011).

23. Watanabe, S. et al. Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis. Microbiology 153, 3044–3054 (2007).

24. Watanabe, S., Kodaki, T. & Makino, K. Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc. J. Biol. Chem. 280, 10340–10349 (2005).

25. Nakata, E., Dinh, H., Ngo, T. A., Saimura, M. & Morii, T. A modular zinc finger adaptor accelerates the covalent linkage of proteins at specific locations on DNA nanoscaffolds. Chem. Commun. 51, 1016–1019 (2015).

26. Ngo, T. A., Nakata, E., Saimura, M. & Morii, T. Spatially organized enzymes drive cofactor-coupled cascade reactions. J. Am. Chem. Soc. 138, 3012–3021 (2016).

27. Nguyen, T. M., Nakata, E., Saimura, M., Dinh, H. & Morii, T. Design of modular protein tags for orthogonal covalent bond formation at specific DNA sequences. J. Am. Chem. Soc. 139, 8487–8496 (2017).

28. Nguyen, T. M. et al. Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics. Chem. Sci. 10, 9315–9325 (2019).

29. Ellenberger, T. E., Brandl, C. J., Struhl, K. & Harrison, S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted α helices: crystal structure of the protein-DNA complex. Cell 71, 1223–1237 (1992).

30. England, C. G., Luo, H. & Cai, W. HaloTag technology: a versatile platform for biomedical applications. Bioconjug. Chem. 26, 975–986 (2015).

31. Eriksson, T., Börjesson, J. & Tjerneld, F. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme Microb. Technol. 31, 353–364 (2002).

32. Yang, B. & Wyman, C. E. BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechnol. Bioeng. 94, 611–617 (2006).

33. Whitaker, J. E., Haugland, R. P. & Prendergast, F. G. Spectral and photophysical studies of benzo[c]xanthene dyes: dual emission pH sensors. Anal. Biochem. 194, 330–344 (1991).

34. Nakata, E. et al. A newly designed cell-permeable SNARF derivative as an effective intracellular pH indicator. Chem. Commun. 46, 3526–3528 (2010).

35. Nakata, E. et al. A novel strategy to design latent ratiometric fluorescent pH probes based on self-assembled SNARF derivatives. RSC Adv. 4, 348–357 (2014).

36. Chen, X., Zhou, Y., Peng, X., & Yoon, J. Fluorescent and colorimetric probes for detection of thiols. Chem. Soc. Rev. 39, 2120-2135 (2010).

37. Langmuir, M. E., Yang, J. R., Moussa, A. M., Laura, R., & LeCompte, K. A. New naphthopyranone based fluorescent thiol probes. Tetrahedron Lett. 36, 3989- 3992 (1995).

38 Torrie, G. M., Kusalik, P. G. & Patey, G. N. Molecular solvent model for an electrical double layer: reference hypernetted-chain results for ion behavior at infinite dilution. J. Chem. Phys. 89, 3285–3294 (1988).

39. Bérard, D. R., Kinoshita, M., Cann, N. M. & Patey, G. N. Structure of the metalaqueous electrolyte solution interface. J. Chem. Phys. 107, 4719–4728 (1997).

40. Kinoshita, M. Water structure and phase transition near a surface. J. Solution Chem. 33, 661–687 (2004).

41. Kratzer, R. et al. Enzyme identification and development of a whole-cell biotransformation for asymmetric reduction of o-chloroacetophenone. Biotechnol. Bioeng. 108, 797–803 (2011).

42. Kratzer, R., Wilson, D. K. & Nidetzky, B. Catalytic mechanism and substrate selectivity of aldo-keto reductases: insights from structure-function studies of Candida tenuis xylose reductase. IUBMB Life 58, 499–507 (2006).

43. Drummond, C., Pérez-Fuentes, L. & Bastos-González, D. Can polyoxometalates be considered as superchaotropic ions? J. Phys. Chem. C 123, 28744–28752 (2019).

44. Collins, K. D. Ions from the Hofmeister series and osmolytes: efects on proteins in solution and in the crystallization process. Methods 34, 300–311 (2004).

45. Verduyn, C. et al. Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis. Biochem. J. 226, 669–677 (1985).

46. Ehrensberger, A. H., Elling, R. A. & Wilson, D. K. Structure-guided engineering of xylitol dehydrogenase cosubstrate specificity. Structure 14, 567–575 (2006).

47. Ngo, T. A., Nakata, E., Saimura, M., Kodaki, T. & Morii, T. A protein adaptor to locate a functional protein dimer on molecular switchboard. Methods 67, 142– 150 (2014).

48. Douglas, S. M. et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001–5006 (2009).

49. Amir, Y. et al. Universal computing by DNA origami robots in a living animal. Nat. Nanotechnol. 9, 353–357 (2014).

50. Han, J. & Burgess, K. Fluorescent indicators for intracellular pH. Chem. Rev. 110, 2709–2728 (2010).

51. Nguyen, T. & Francis, M. B. Practical synthetic route to functionalized rhodamine dyes. Org. Lett. 5, 3245–3248 (2003).

52. Benjaminsen, R. V. et al. Evaluating nanoparticle sensor design for intracellular pH measurements. ACS Nano 5, 5864–5873 (2011).

Chapter3

1. Agapakis, C. M., Boyle, P. M. & Silver, P. A. Natural strategies for the spatial optimization of metabolism in synthetic biology. Nat. Chem. Biol. 8, 527–535 (2012).

2. Küchler, A., Yoshimoto, M., Luginbühl, S., Mavelli, F. & Walde, P. Enzymatic reactions in confined environments. Nat. Nanotechnol. 11, 409–420 (2016).

3. Conrado, R. J., Varner, J. D. & DeLisa, M. P. Engineering the spatial organization of metabolic enzymes: mimicking nature’s synergy. Curr. Opin. Biotechnol. 19, 492–499 (2008).

4. Bonacci, W. et al. Modularity of a carbon-fixing protein organelle. Proc. Natl. Acad. Sci. 109, 478–483 (2012).

5. Gou, M., Ran, X., Martin, D. W. & Liu, C. J. The scaffold proteins of lignin biosynthetic cytochrome P450 enzymes. Nat. Plants 4, 299–310 (2018).

6. Liu, L. N. Distribution and dynamics of electron transport complexes in cyanobacterial thylakoid membranes. Biochim. Biophys. Acta-Bioenerg. 1857, 256–265 (2016).

7. Brasch, M. et al. Assembling enzymatic cascade pathways inside virus-based nanocages using dual-tasking nucleic acid tags. J. Am. Chem. Soc. 139, 1512– 1519 (2017).

8. Walde, P. & Ichikawa, S. Enzymes inside lipid vesicles: preparation, reactivity and applications. Biomol. Eng. 18, 143–177 (2001).

9. Klermund, L., Poschenrieder, S. T. & Castiglione, K. Biocatalysis in polymersomes: improving multienzyme cascades with incompatible reaction steps by compartmentalization. ACS Catal. 7, 3900–3904 (2017).

10. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

11. Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).

12. Hong, F., Zhang, F., Liu, Y. & Yan, H. DNA origami: scaffolds for creating higher order structures. Chem. Rev. 117, (2017).

13. Simmel, F. C., Yurke, B. & Singh, H. R. Principles and applications of nucleic acid strand displacement reactions. Chem. Rev. 119, 6326-6369 (2019).

14. Rangel, A. E., Hariri, A. A., Eisenstein, M. & Soh, H. T. Engineering aptamer switches for multifunctional stimulus-responsive nanosystems. Adv. Mater. (2020).

15. Turek, V. A. et al. Thermo-responsive actuation of a DNA origami flexor. Adv. Funct. Mater. 28, 1–7 (2018).

16. Kim, S. H. et al. Reversible regulation of enzyme activity by pH-responsive encapsulation in DNA nanocages. ACS Nano 11, 9352–9359 (2017).

17. Deluca, M., Shi, Z., Castro, C. E. & Arya, G. Dynamic DNA nanotechnology: toward functional nanoscale devices. Nanoscale Horizons 5, 182–201 (2020).

18. Juul, S. et al. Temperature-controlled encapsulation and release of an active enzyme in the cavity of a self-assembled DNA nanocage. ACS Nano 7, 9724– 9734 (2013).

19. Ijäs, H., Hakaste, I., Shen, B., Kostiainen, M. A. & Linko, V. Reconfigurable DNA origami nanocapsule for pH-controlled encapsulation and display of cargo. ACS Nano 13, 5959-5967 (2019).

20. Marras, A. E. et al. Cation-activated avidity for rapid reconfiguration of DNA nanodevices. ACS Nano 12, 9484–9494 (2018).

21. Grossi, G., Dalgaard Ebbesen Jepsen, M., Kjems, J. & Andersen, E. S. Control of enzyme reactions by a reconfigurable DNA nanovault. Nat. Commun. 8, (2017).

22. Xin, L., Zhou, C., Yang, Z. & Liu, D. Regulation of an enzyme cascade reaction by a DNA machine. Small 9, 3088–3091 (2013).

23. Rajendran, A., Nakata, E., Nakano, S. & Morii, T. Nucleic-acid-templated enzyme cascades. ChemBioChem 18, 696–716 (2017).

24. Douglas, S. M., Bachelet, I. & Church, G. M. A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831–834 (2012).

25. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–16 (2008).

26. Markegard, C. B., Gallivan, C. P., Cheng, D. D. & Nguyen, H. D. Effects of concentration and temperature on DNA hybridization by two closely related sequences via large-scale coarse-grained simulations. J. Phys. Chem. B 120, 7795–7806 (2016).

27. Groeer, S. & Walther, A. Switchable supracolloidal 3D DNA origami nanotubes mediated through fuel/antifuel reactions. Nanoscale 12, 16995–17004 (2020).

28. Watanabe, S., Kodaki, T. & Makino, K. Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc. J. Biol. Chem. 280, 10340–10349 (2005).

29. Nakata, E., Dinh, H., Ngo, T. A., Saimura, M. & Morii, T. A modular zinc finger adaptor accelerates the covalent linkage of proteins at specific locations on DNA nanoscaffolds. Chem. Commun. 51, 1016–1019 (2015).

30. Ngo, T. A., Nakata, E., Saimura, M. & Morii, T. Spatially organized enzymes drive cofactor-coupled cascade reactions. J. Am. Chem. Soc. 138, 3012–3021 (2016).

31. Nguyen, T. M., Nakata, E., Saimura, M., Dinh, H. & Morii, T. Design of modular protein tags for orthogonal covalent bond formation at specific DNA sequences. J. Am. Chem. Soc. 139, 8487–8496 (2017).

32. Nguyen, T. M. et al. Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics. Chem. Sci. 10, 9315–9325 (2019).

33. Ellenberger, T. E., Brandl, C. J., Struhl, K. & Harrison, S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted α helices: crystal structure of the protein-DNA complex. Cell 71, 1223–1237 (1992).

34. England, C. G., Luo, H. & Cai, W. HaloTag technology: a versatile platform for biomedical applications. Bioconjug. Chem. 26, 975–986 (2015).

35. Lin, P. et al. Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes. Chem. Commun. 57, 3925–3928 (2021).

36. Zhao, Z. et al. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion. Nat. Commun. 7, (2016).

37. Timm, C. & Niemeyer, C. M. Assembly and purification of enzymefunctionalized DNA origami structures. Angew. Chem., Int. Ed. 54, 6745–6750 (2015).

38. Lin, J. L. & Wheeldon, I. Kinetic enhancements in DNA-enzyme nanostructures mimic the sabatier principle. ACS Catal. 3, 560–564 (2013).

39. Zhang, Y., Tsitkov, S. & Hess, H. Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade. Nat. Commun. 7, (2016).

40. Xiong, Y., Huang, J., Wang, S. T., Zafar, S. & Gang, O. Local environment affects the activity of enzymes on a 3D molecular scaffold. ACS Nano 14, 14646–14654 (2020).

41. Rudiuk, S., Venancio-Marques, A. & Baigl, D. Enhancement and modulation of enzymatic activity through higher-order structural changes of giant DNA-Protein multibranch conjugates. Angew. Chem., Int. Ed. 51, 12694–12698 (2012).

42. Huang, J. et al. Arranging small molecules with subnanometer precision on DNA origami substrates for the single-molecule investigation of protein-ligand interactions. Small Structures 1 : 2000038 (2020).

43. Zhang, P. et al. Quantitative measurement of spatial effects of DNA origami on molecular binding reactions detected using atomic force microscopy. ACS Appl. Mater. Interfaces 11, 21973-21981 (2019).

44. Dinh, H. et al. Enhanced enzymatic activity exerted by a packed assembly of a single type of enzyme. Chem. Sci. 11, 9088–9100 (2020).

45. Douglas, S. M. et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001–5006 (2009).

46. Amir, Y. et al. Universal computing by DNA origami robots in a living animal. Nat. Nanotechnol. 9, 353–357 (2014).

Chapter4

1. Wheeldon, I. et al. Substrate channelling as an approach to cascade reactions. Nat. Chem. 8, 299–309 (2016).

2. Conrado, R. J., Varner, J. D. & DeLisa, M. P. Engineering the spatial organization of metabolic enzymes: mimicking nature’s synergy. Curr. Opin. Biotechnol. 19, 492–499 (2008).

3. Miles, E. W., Rhee, S. & Davies, D. R. The molecular basis of substrate channeling. J. Biol. Chem. 274, 12193–12196 (1999).

4. Wu, F. & Minteer, S. Krebs cycle metabolon: structural evidence of substrate channeling revealed by cross-linking and mass spectrometry. Angew. Chemie., Int. Ed. 54, 1851–1854 (2015).

5. Bonacci, W. et al. Modularity of a carbon-fixing protein organelle. Proc. Natl. Acad. Sci. 109, 478–483 (2012).

6. Dueber, J. E. et al. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759 (2009).

7. Chen, L., Mulchandani, A. & Ge, X. Spore-displayed enzyme cascade with tunable stoichiometry. Biotechnol. Prog. 33, 383–389 (2017).

8. Xin, L., Zhou, C., Yang, Z. & Liu, D. Regulation of an enzyme cascade reaction by a DNA machine. Small 9, 3088–3091 (2013).

9. Wilner, O. I. et al. Enzyme cascades activated on topologically programmed DNA scaffolds. Nat. Nanotechnol. 4, 249–254 (2009).

10. Fu, J., Liu, M., Liu, Y., Woodbury, N. W. & Yan, H. Interenzyme substrate diffusion for an enzyme cascade organized on spatially addressable DNA nanostructures. J. Am. Chem. Soc. 134, 5516–5519 (2012).

11. Zhang, Y., Tsitkov, S. & Hess, H. Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade. Nat. Commun. 7, (2016).

12. Hinzpeter, F., Gerland, U. & Tostevin, F. Optimal compartmentalization strategies for metabolic microcompartments. Biophys. J. 112, 767–779 (2017).

13. Ngo, T. A., Nakata, E., Saimura, M. & Morii, T. Spatially organized enzymes drive cofactor-coupled cascade reactions. J. Am. Chem. Soc. 138, 3012–3021 (2016).

14. Douglas, S. M., Bachelet, I. & Church, G. M. A logic-gated nanorobot for targeted transport of molecular payloads. Science. 335, 831–834 (2012).

15. Zhang, Y. & Hess, H. Toward rational design of high-efficiency enzyme cascades. ACS Catal. 7, 6018–6027 (2017).

16. Tsitkov, S., Pesenti, T., Palacci, H., Blanchet, J. & Hess, H. Queueing theorybased perspective of the kinetics of ‘channeled’ enzyme cascade reactions. ACS Catal. 8, 10721–10731 (2018).

17. Watanabe, S., Kodaki, T. & Makino, K. Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc. J. Biol. Chem. 280, 10340–10349 (2005).

18. Pival, S. L., Birner-Gruenberger, R., Krump, C. & Nidetzky, B. D-Xylulose kinase from Saccharomyces cerevisiae: isolation and characterization of the highly unstable enzyme, recombinantly produced in Escherichia coli. Protein Expr. Purif. 79, 223–230 (2011).

19. Nakata, E., Dinh, H., Ngo, T. A., Saimura, M. & Morii, T. A modular zinc finger adaptor accelerates the covalent linkage of proteins at specific locations on DNA nanoscaffolds. Chem. Commun. 51, 1016–1019 (2015).

20. Nguyen, T. M., Nakata, E., Saimura, M., Dinh, H. & Morii, T. Design of modular protein tags for orthogonal covalent bond formation at specific DNA sequences. J. Am. Chem. Soc. 139, 8487–8496 (2017).

21. Nguyen, T. M. et al. Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics. Chem. Sci. 10, 9315–9325 (2019).

22. Lin, P. et al. Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes. Chem. Commun. 57, 3925–3928 (2021).

23. Greisman, H. A. & Pabo, C. O. A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites. Science 275, 657–661 (1997).

24. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

25. Burton, R. M. & Kaplan, N. O. The reaction of reduced pyridine nucleotides with acid. Arch. Biochem. Biophys. 101, 150–159 (1963).

26. Wu, J. T., Wu, L. H. & Knight, J. A. Stability of NADPH: effect of various factors on the kinetics of degradation. Clin. Chem. 32, 314–319 (1986).

27. Walsh, K. A., Daniel, R. M. & Morgan, H. W. A soluble NADH dehydrogenase (NADH: ferricyanide oxidoreductase) from Thermus aquaticus strain T351. Biochem. J. 209, 427–33 (1983).

28. Rover, L. et al. Study of NADH stability using ultraviolet-visible spectrophotometric analysis and factorial design. Anal. Biochem. 260, 50–55 (1998).

29. Douglas, S. M. et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001–5006 (2009).

30 Amir, Y., Ben-Ishay, E., Levner, D., Ittah, S., Abu-Horowitz, A., & Bachelet, I. Universal computing by DNA origami robots in a living animal. Nat. Nanotechnol. 9, 353-357 (2014).

31. Erickson, H. P. Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biol. Proced. Online 11, 32–51 (2009).

Chapter5

1. Agapakis, C. M., Boyle, P. M. & Silver, P. A. Natural strategies for the spatial optimization of metabolism in synthetic biology. Nat. Chem. Biol. 8, 527–535 (2012).

2. Pareek, V., Tian, H., Winograd, N. & Benkovic, S. J. Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells. Science 368, 283–290 (2020).

3. Bulutoglu, B., Garcia, K. E., Wu, F., Minteer, S. D. & Banta, S. Direct evidence for metabolon formation and substrate channeling in recombinant TCA cycle enzymes. ACS Chem. Biol. 11, 2847–2853 (2016).

4. Sweetlove, L. J. & Fernie, A. R. The role of dynamic enzyme assemblies and substrate channelling in metabolic regulation. Nat. Commun. 9, (2018).

5. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

6. Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).

7. Saccà, B. et al. Orthogonal protein decoration of DNA origami. Angew. Chem., Int. Ed. 49, 9378–9383 (2010).

8. Yurke, B., Turber, A. J., Jr, A. P. M., Simmel, F. C. & Neumann, J. L. A DNAfuelled molecular machine made of DNA. Nature 406, 605–608 (2000).

9. Grossi, G., Dalgaard Ebbesen Jepsen, M., Kjems, J. & Andersen, E. S. Control of enzyme reactions by a reconfigurable DNA nanovault. Nat. Commun. 8, (2017).

10. Andersen, E. S. et al. Self-assembly of a nanoscale DNA box with a controllable lid. Nature 459, 73–76 (2009).

11. Zadegan, R. M. et al. Construction of a 4 Zeptoliters switchable 3D DNA box origami. ACS Nano 6, 10050–10053 (2012).

12. Douglas, S. M., Bachelet, I. & Church, G. M. A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831–834 (2012).

13. Li, S. et al. A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivo. Nat. Biotechnol. 36, 258–264 (2018).

14. Marras, A. E. et al. Cation-activated avidity for rapid reconfiguration of DNA nanodevices. ACS Nano 12, 9484–9494 (2018).

15. Kuzuya, A., Sakai, Y., Yamazaki, T., Xu, Y. & Komiyama, M. Nanomechanical DNA origami ‘single-molecule beacons’ directly imaged by atomic force microscopy. Nat. Commun. 2, (2011).

16. Ijäs, H., Hakaste, I., Shen, B., Kostiainen, M. A. & Linko, V. Reconfigurable DNA origami nanocapsule for pH-controlled encapsulation and display of cargo. ACS Nano 13, 5959–5967 (2019).

17. Kim, S. H. et al. Reversible regulation of enzyme activity by pH-responsive encapsulation in DNA nanocages. ACS Nano 11, 9352–9359 (2017).

18. Turek, V. A. et al. Thermo-responsive actuation of a DNA origami flexor. Adv. Funct. Mater. 28, 1–7 (2018).

19. Juul, S. et al. Temperature-controlled encapsulation and release of an active enzyme in the cavity of a self-assembled DNA nanocage. ACS Nano 7, 9724– 9734 (2013).

20. Kohman, R. E. & Han, X. Light sensitization of DNA nanostructures via incorporation of photo-cleavable spacers. Chem. Commun. 51, 5747–5750 (2015).

21. Feng, Y., Tohgasaki, T., Shitomi, Y., Sugiyama, H. & Endo, M. A photocaged DNA nanocapsule for delivery and manipulation in cells. Methods Enzymol. 641, 329-342 (2020).

22. Kopperger, E. et al. A self-assembled nanoscale robotic arm controlled by electric fields. Science 359, 296–301 (2018).

23. Linko, V. et al. DNA-based enzyme reactors and systems. Nanomaterials 6, 139 (2016).

24. Lin, P. et al. Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes. Chem. Commun. 57, 3925–3928 (2021).

25. Linko, V., Eerikäinen, M. & Kostiainen, M. A. A modular DNA origami-based enzyme cascade nanoreactor. Chem. Commun. 51, 5351–5354 (2015).

26. Fu, Y. et al. Single-step rapid assembly of DNA origami nanostructures for addressable nanoscale bioreactors. J. Am. Chem. Soc. 135, 696–702 (2013).

27. Zhao, Z. et al. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion. Nat. Commun. 7, (2016).

28. Zhang, D. Y. & Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131, 17303–17314 (2009).

29. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).

30. Watanabe, S. et al. Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis. Microbiology 153, 3044–3054 (2007).

31. Watanabe, S., Kodaki, T. & Makino, K. Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc. J. Biol. Chem. 280, 10340–10349 (2005).

32. Nakata, E., Dinh, H., Ngo, T. A., Saimura, M. & Morii, T. A modular zinc finger adaptor accelerates the covalent linkage of proteins at specific locations on DNA nanoscaffolds. Chem. Commun. 51, 1016–1019 (2015).

33. Ngo, T. A., Nakata, E., Saimura, M. & Morii, T. Spatially organized enzymes drive cofactor-coupled cascade reactions. J. Am. Chem. Soc. 138, 3012–3021 (2016).

34. Nguyen, T. M., Nakata, E., Saimura, M., Dinh, H. & Morii, T. Design of modular protein tags for orthogonal covalent bond formation at specific DNA sequences. J. Am. Chem. Soc. 139, 8487–8496 (2017).

35. Nguyen, T. M. et al. Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics. Chem. Sci. 10, 9315–9325 (2019).

36. Idan, O. & Hess, H. Origins of activity enhancement in enzyme cascades on scaffolds. ACS Nano 7, 8658–8665 (2013).

37. Chado, G. R., Stoykovich, M. P. & Kaar, J. L. Role of dimension and spatial arrangement on the activity of biocatalytic cascade reactions on scaffolds. ACS Catal. 6, 5161–5169 (2016).

38. Fu, J., Liu, M., Liu, Y., Woodbury, N. W. & Yan, H. Interenzyme substrate diffusion for an enzyme cascade organized on spatially addressable DNA nanostructures. J. Am. Chem. Soc. 134, 5516–5519 (2012).

39. Lim, S., Kim, J., Kim, Y., Xu, D. & Clark, D. S. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chem. Commun. 56, 4950– 4953 (2020).

40. Cao, Y. et al. Investigating the origin of high efficiency in confined multienzyme catalysis. Nanoscale 11, 22108–22117 (2019).

41. Lin, J. L. & Wheeldon, I. Kinetic enhancements in DNA-enzyme nanostructures mimic the sabatier principle. ACS Catal. 3, 560–564 (2013).

42. Zhang, Y., Tsitkov, S. & Hess, H. Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade. Nat. Commun. 7, (2016).

43. Zhang, Y. & Hess, H. Toward rational design of high-efficiency enzyme cascades. ACS Catal. 7, 6018–6027 (2017).

44. Timm, C. & Niemeyer, C. M. Assembly and purification of enzymefunctionalized DNA origami structures. Angew. Chem., Int. Ed. 54, 6745–6750 (2015).

45. Rudiuk, S., Venancio-Marques, A. & Baigl, D. Enhancement and modulation of enzymatic activity through higher-order structural changes of giant DNA-protein multibranch conjugates. Angew. Chem., Int. Ed. 51, 12694–12698 (2012).

46. Wu, N. et al. Molecular threading and tunable molecular recognition on DNA origami nanostructures. J. Am. Chem. Soc. 135, 12172–12175 (2013).

47. Douglas, S. M. et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001–5006 (2009).

48. Amir, Y. et al. Universal computing by DNA origami robots in a living animal. Nat. Nanotechnol. 9, 353–357 (2014).

Chapter6

1. Ngo, T. A., Nakata, E., Saimura, M. & Morii, T. Spatially organized enzymes drive cofactor-coupled cascade reactions. J. Am. Chem. Soc. 138, 3012–3021 (2016).

参考文献をもっと見る