Masayuki Endo, PhD, is a project professor at Kansai University and a guest professor at Kyoto University, Japan. He received his PhD from the Department of Chemistry and Biotechnology, The University of Tokyo in 1997. Prof. Endo's research work involves DNA nanotechnology and single-molecule analysis.

List of Contributors xiii

Preface xvii

1 DNA Origami Technology: Achievements in the Initial 10 Years 1
Masayuki Endo

1.1 Introduction 1

1.1.1 DNA Nanotechnology Before the Emergence of DNA Origami 3

1.2 Two- Dimensional DNA Origami 3

1.3 Programmed Arrangement of Multiple DNA Origami Components 6

1.4 Three- Dimensional DNA Origami Structures 9

1.5 Modification and Functionalization of 2D DNA Origami Structures 11

1.5.1 Selective Placement of Functional Nanomaterials 11

1.5.2 Selective Placement of Functional Molecules and Proteins via Ligands 13

1.5.3 Distance- Controlled Enzyme Reactions and Photoreactions 13

1.6 Single- Molecule Detection and Sensing using DNA Origami Structures 14

1.6.1 Single- Molecule RNA Detection 14

1.6.2 Single- Molecule Detection of Chemical Reactions 14

1.6.3 Single- Molecule Detection using Mechanical DNA Origami 14

1.6.4 Single- Molecule Sensing using Mechanical DNA Origami 14

1.7 Application to Single Biomolecule AFM Imaging 16

1.7.1 High- Speed AFM- Based Observation of Biomolecules 16

1.7.2 Visualization of DNA Structural Changes in the DNA Nanospace 18

1.7.3 Visualization of the Reaction Events of Enzymes and Proteins in the DNA Nanospace 18

1.8 Single- Molecule Fluorescence Studies 19

1.8.1 Nanoscopic Ruler for Single- Molecule Imaging 19

1.8.2 Kinetics of Binding and Unbinding Events and DNA- PAINT 21

1.8.3 DNA Barcode Imaged by DNA- PAINT 21

1.9 DNA Molecular Machines 22

1.9.1 DNA Assembly Line Constructed on the DNA Origami 22

1.9.2 DNA Spider System Constructed on the DNA Origami 22

1.9.3 DNA Motor System Constructed on the DNA Origami 24

1.10 Selective Incorporation of Nanomaterials and the Applications 24

1.10.1 DNA Origami Plasmonic Structure with Chirality 24

1.10.2 Surface- Enhanced Fluorescence by Gold Nanoparticles and DNA Origami Structure 26

1.10.3 Placement of DNA Origami onto a Fabricated Solid Surface 26

1.11 Dynamic DNA Origami Structures Responsive to External Stimuli 27

1.11.1 DNA Origami Structures Responsive to External Stimuli 27

1.11.2 Stimuli- Responsive DNA Origami Plasmonic Structures 27

1.11.3 Photo- Controlled DNA Origami Plasmonic Structures 27

1.12 Conjugation of DNA Origami to Lipid 29

1.12.1 DNA Origami Channel with Gating 29

1.12.2 DNA Origami Templated Synthesis of Liposomes 29

1.13 DNA Origami for Biological Applications 29

1.13.1 Introduction of DNA Origami into Cells and Functional Expression 29

1.13.2 Drug Release Using the Properties Characteristic for DNA Origami 31

1.13.3 DNA Origami Structures Coated with Lipids and Polymers 32

1.13.4 Nanorobot with Dynamic Mechanism 32

1.13.5 Nanorobot Targeting Tumor In Vivo 32

1.14 Conclusions 33

References 34

2 Wireframe DNA Origami and Its Application as Tools for Molecular Force Generation 41
Marco Lolaico and Björn Högberg

2.1 Introduction 41

2.2 Pre- Origami Wireframe DNA Nanostructures 42

2.3 Hierarchical DNA Origami Wireframe 43

2.4 Entire DNA Origami Design 45

2.5 DNA Origami Wireframe as Tools for Molecular Force Application 50

2.5.1 Introduction 50

2.5.2 Results and Discussion 51

2.6 Conclusions 54

2.6.1 Materials and Methods 54

References 55

3 Capturing Structural Switching and Self- Assembly Events Using High- Speed Atomic Force Microscopy 59
Yuki Suzuki

3.1 Introduction 59

3.2 DNA Origami Nanomachines 60

3.3 Ion- Responsive Mechanical DNA Origami Devices 60

3.4 Photoresponsive Devices 62

3.5 Two- Dimensional Self- Assembly Processes 64

3.6 Sequential Self- Assembly 66

3.7 Photostimulated Assembly and Disassembly 67

3.8 Conclusions and Perspectives 69

References 69

4 Advancement of Computer- Aided Design Software and Simulation Tools for Nucleic Acid Nanostructures and DNA Origami 75
Ibuki Kawamata

4.1 Introduction 75

4.2 General- Purpose Software 76

4.3 Software for Designing Small DNA Nanostructures 78

4.4 Software for Designing DNA Origami 81

4.5 Software for Designing RNA Nanostructures 84

4.6 Software for Designing Base Sequence 84

4.7 Software for Simulating Nucleic Acid Nanostructures 85

4.8 Summary and Future Perspective 86

References 87

5 Dynamic and Mechanical Applications of DNA Nanostructures in Biophysics 101
Melika Shahhosseini, Anjelica Kucinic, Peter Beshay, Wolfgang Pfeifer, and Carlos Castro

5.1 Introduction 101

5.1.1 What Makes DNA a Good Material for Dynamic Applications 101

5.1.2 Rupture Forces 103

5.2 Applications 105

5.2.1 Force Spectroscopy 105

5.2.1.1 Utilizing the Stiffness of DNA for Force Spectroscopy 105

5.2.1.2 Applications that Utilize Rupture Forces 107

5.2.2 DNA Devices that Probe and Control DNA-DNA Interactions 108

5.2.2.1 Detection 108

5.2.2.2 Modulation 111

5.2.3 DNA Devices that Respond to Biomolecules 111

5.2.4 DNA Devices to Study Biological Molecular Motors 116

5.2.5 DNA Walkers 116

5.2.6 DNA Computing 119

5.3 Tools for Quantifying DNA Devices and their Functions 120

5.4 Modeling and Analysis 123

5.5 Conclusion 124

References 124

6 Plasmonic Nanostructures Assembled by DNA Origami 135
Sergio Kogikoski, Jr, Anushree Dutta, and Ilko Bald

6.1 Introduction 135

6.2 Optical Properties of the DNA Origami- Based Plasmonic Nanostructures 135

6.3 Nanoparticle Functionalization with DNA 138

6.4 DNA Origami- Based Plasmonic Assemblies 140

6.5 Surface- Enhanced Raman Scattering (SERS) and Other Plasmonic Effects 143

6.6 Conclusion 152

Acknowledgments 152

References 152

7 Assembly of Nanoparticle Superlattices Using DNA Origami as a Template 155
Sofia Julin, Petteri Piskunen, Mauri A. Kostiainen, and Veikko Linko

7.1 Introduction 155

7.2 Gold Nanoparticles 156

7.2.1 Oligonucleotide- Modified AuNPs 156

7.2.2 Cationic AuNPs 158

7.3 Formation of DNA Origami- Assisted Superlattices 158

7.3.1 Superlattices Formed by Oligonucleotide- Functionalized AuNPs 159

7.3.2 Superlattice Formed by Cationic AuNPs 160

7.4 Characterization of Assemblies 160

7.4.1 Electron Microscopy 161

7.4.2 Small- Angle X- ray Scattering 161

7.5 Conclusions and Future Perspectives 162

Acknowledgments 164

References 164

8 Mechanics of DNA Origami Nanoassemblies 167
Deepak Karna, Jiahao Ji, and Hanbin Mao

8.1 Introduction 167

8.2 Analytical Tools to Investigate Mechanical Properties of Nanoassemblies 168

8.2.1 Optical Tweezers 168

8.2.2 Magnetic Tweezers 169

8.2.3 Atomic Force Microscopy (AFM) 169

8.3 Mechanical Strength of DNA Origami Structures 171

8.4 Applications of Origami Nanostructures by Exploiting their Mechanical Strength 173

8.5 Mechanochemical Properties of DNA Origami 175

8.6 Conclusions 177

References 177

9 3D DNA Origami as Single- Molecule Biophysical Tools for Dissecting Molecular Motor Functions 181
Mitsuhiro Iwaki

9.1 Introduction 181

9.2 DNA Origami Nanospring 181

9.2.1 Design of DNA Origami Nanospring 181

9.2.2 Nanospring Mechanical Properties 182

9.2.3 Application to a Myosin VI Processive Motor 183

9.3 DNA Origami Thick Filament Mimicking Muscle Structure 187

9.3.1 Mystery of Muscle Contraction 187

9.3.2 Design of a DNA Origami- Based Thick Filament 188

9.3.3 High- speed AFM Observation of Force Generation by Myosin 189

9.3.4 High- Speed Darkfield Imaging of Force Generation by Myosin 189

9.4 Perspective 193

References 193

10 Switchable DNA Origami Nanostructures and Their Applications 197
Jianbang Wang, Michael P. O'Hagan, Verena Wulf, and Itamar Willner

10.1 Introduction 197

10.2 Switchable Machines Constructed from DNA Origami Scaffolds 198

10.2.1 Chemical Triggers for Origami Scaffolds 198

10.2.1.1 Triggering Origami Devices with Strand Displacement Reactions 198

10.2.1.2 Triggering Origami with Ion Concentration 200

10.2.1.3 Triggering Origami with Molecular Species 202

10.2.2 Physical Triggers for Origami Scaffolds 204

10.2.2.1 Triggering Origami with Temperature 204

10.2.2.2 Triggering Origami with Electric Fields 206

10.2.2.3 Triggering Origami with Magnetic Fields 206

10.2.2.4 Triggering Origami with Light 208

10.3 DNA Origami Scaffolds for Defined Mechanical Operations 210

10.3.1 Origami Scaffolds that Dictate the Motility of Elements 212

10.3.2 Engineering Mechanical Functions of Origami Tiles 218

10.4 Switchable Interconnected 2D Origami Assemblies 218

10.5 Dynamic Triggered Switching of Origami for Controlled Release 223

10.6 Switchable Plasmonic Phenomena with DNA Origami Scaffolds 227

10.7 Origami- Guided Organization of Nanoparticles and Proteins 234

10.8 Conclusions and Perspectives 238

References 239

11 The Effect of DNA Boundaries on Enzymatic Reactions 241
Richard Kosinski and Barbara Saccà

11.1 Introduction 241

11.2 DNA- Scaffolded Single Enzymes 242

11.3 DNA- Scaffolded Enzyme Cascades 247

11.4 On the Proximity Model and Other Hypotheses 250

11.5 Conclusions 254

Acknowledgments 256

References 256

12 The Methods to Assemble Functional Proteins on DNA Scaffold and their Applications 261
Eiji Nakata, Shiwei Zhang, Huyen Dinh, Peng Lin, and Takashi Morii

12.1 Introduction 261

12.2 Overview of the Methods for Arranging Proteins on DNA Scaffolds 262

12.2.1 Reversible Conjugation between Protein and DNA 263

12.2.1.1 Biotin- Avidin 264

12.2.1.2 Antibody- Antigen 264

12.2.1.3 Ni- NTA- Hexahistidine 266

12.2.1.4 Aptamers 266

12.2.1.5 Apo- Protein Reconstitution by the Prosthetic Group 266

12.2.2 Irreversible Conjugation between Protein and DNA 266

12.2.2.1 Chemical Crosslinking of Protein and DNA via Cross- Linker 267

12.2.2.2 Crosslinking of Genetically Fused Protein with Chemically Modified DNA 267

12.2.2.3 Covalent Conjugation of Genetically Modified Proteins to Unmodified DNA 269

12.2.2.4 Applications of the Enzyme Assembled DNA Scaffolds 269

12.3 DNA- Binding Adaptor for Assembling Proteins on DNA Scaffold and its Application 270

12.3.1 DNA- Binding Adaptor for Reversible Assembly of Proteins via Noncovalent Interactions 270

12.3.2 Modular Adaptors for Covalent Conjugation of Genetically Modified Proteins to Chemically Modified DNA 272

12.3.3 Application of DNA-...