DNA and RNA Origami: Methods and Protocols (Methods in Molecular Biology, 2639)

✍ Scribed by Julián Valero (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 351
• Edition: 1st ed. 2023
• Category: Library

✦ Synopsis

This volume detials diverse methodological approaches on the assembly and applications of DNA origami assemblies. Chapters guide readers through different synthetic and computational methods, isolation and structural characterization of 2D and 3D DNA origami nanoarchitectures,  nanophotonics, drug delivery, biophysics, and synthetic biology.Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

 

Authoritative and cutting-edge, DNA and RNA Origami: Methods and Protocols aims to serve as a guideline describing the current state-of-the-art assembly methodologies and applications of DNA origami nanostructures.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Background and Design of Nucleic Acid Origami
Chapter 1: DNA Origami: Recent Progress and Applications
1 Introduction
2 History and Development
2.1 Structural DNA Origami
2.2 Dynamic DNA Origami
3 Applications
3.1 Prototype Applications
3.2 Expanding the Toolbox of Applications
4 Conclusion
References
Chapter 2: Design, Assembly, and Function of DNA Origami Mechanisms
1 Introduction
2 Materials
2.1 DNA Origami Self-Assembly
2.2 Purification
2.2.1 Gel Electrophoresis
2.2.2 Centrifugation in Polyethylene Glycol (PEG Solution)
2.2.3 Molecular Weight Cutoff (MWCO) Centrifugal Filters
2.3 Transmission Electron Microscopy (TEM) Imaging and Verification Methods
3 Methods
3.1 DNA Origami Design
3.1.1 Simple Structures
3.1.2 Design of Overhangs (See Note 4)
3.1.3 Design of Dynamic Origami
3.1.4 Design of Actuation Methods
3.1.5 Design of Readout Methods (See Note 12)
3.2 DNA Origami Assembly (See Note 16) (Depending on Applications, See Notes 25, 26, and 27)
3.2.1 Prestocks and Working Stocks and Folding Reactions
3.2.2 DNA Origami Thermal Annealing Protocols (See Note 20)
3.2.3 Rapid Folding
3.3 Purification
3.3.1 Gel Electrophoresis
3.3.2 Polyethylene Glycol (PEG) Purification
3.3.3 Molecular Weight Cutoff (MWCO) Filters
3.4 Imaging and Verification Methods
3.4.1 Grid Preparation for Transmission Electron Microscopy (TEM) (See Notes 43 and 44)
4 Notes
References
Chapter 3: Computer-Aided Design and Production of RNA Origami as Protein Scaffolds and Biosensors
1 Introduction
2 Materials
2.1 Software
2.2 Wet Lab Materials
2.2.1 DNA Template Amplification and Purification
2.2.2 In Vitro Transcription of RNA
2.2.3 Denaturing Acrylamide Gel Electrophoresis
2.2.4 Folding RNA Origami by Heat-Annealing Procedure
2.2.5 Fluorescence Spectroscopy
2.2.6 Atomic Force Microscopy
3 Methods
3.1 Design and Characterization of RNA Origami Programmable Lattices with bKLs
3.1.1 RNA Origami Design
3.1.2 DNA Template Production
3.1.3 RNA Transcription on Mica and AFM Imaging
3.2 Design and Characterization of an RNA Origami-Based FRET Sensor System
3.2.1 Incorporation of Functional Motifs into the Origami
3.2.2 RNA Production and Purification
3.2.3 FRET Measurements
4 Notes
References
Chapter 4: Reconfigurable Two-Dimensional DNA Molecular Arrays
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Reagent Setup
3 Methods
3.1 Folding of DNA Origami Structures
3.2 Purification of DNA Nanostructures
3.3 AFM Imaging of DNA Nanostructures
3.4 TEM Imaging of DNA Nanostructures
3.5 Regulation of DNA Molecular Array Transformation
4 Notes
References
Chapter 5: Two-Dimensional DNA Origami Lattices Assembled on Lipid Bilayer Membranes
1 Introduction
2 Materials
2.1 DNA Origami Structures
2.2 Mica-Supported Lipid Bilayers
2.3 Lipid-Bilayer-Assisted Self-Assembly of 2D DNA Origami Lattices
3 Methods
3.1 DNA Origami Structures
3.1.1 Preparation of DNA Origami Structures
3.1.2 Agarose Gel Electrophoresis
3.1.3 AFM Imaging
3.2 Lipid-Bilayer-Assisted Self-Assembly of DNA Origami Lattices
3.2.1 Preparation of Mica-SLBs
3.2.2 Lipid-Bilayer-Assisted Self-Assembly of DNA Origami Lattices
4 Notes
References
Part II: Molecular Dynamics and Simulations of DNA Origami
Chapter 6: The oxDNA Coarse-Grained Model as a Tool to Simulate DNA Origami
1 Introduction
2 Materials
2.1 Software
2.2 Files
3 Methods
3.1 Conversion to oxDNA Format
3.2 Relaxation of Initial Geometry
3.3 Origami Simulation
3.4 Analysis of a Simulation Trajectory
4 Notes
References
Chapter 7: All-Atom Molecular Dynamics Simulations of Membrane-Spanning DNA Origami Nanopores
1 Introduction
2 Materials
2.1 Software and Online Servers
2.2 Required Files
3 Methods
3.1 Assembling All-Atom Model of DNA Origami Nanopore in Lipid Bilayer Membrane
3.2 Equilibrating the Structure of DNA Origami Nanopores in Lipid Bilayer Membrane (See Note 3)
3.3 Electric Field Simulations
3.4 Calculation of Ionic Current and Lipid Scrambling
4 Notes
References
Part III: Single-Molecule Characterization of DNA Origami
Chapter 8: Single-Molecule Imaging of Enzymatic Reactions on DNA Origami
1 Introduction
2 Materials
2.1 Chemicals
2.2 Buffer Solutions
3 Methods
3.1 DNA-Enzyme Conjugation and Characterization
3.2 Assembly and Characterization of GOx on DNA Origami
3.3 Preparation of Liposome and Supported Lipid Bilayer (SLB)
3.4 GOx and Catalase Anchored on Phospholipid Bilayer
3.5 Imaging of Enzymatic Cascade Reactions
3.6 Fluorescence Recovery After Photobleaching (FRAP) Experiment
3.7 Fluorescence Anisotropy Experiment
3.8 Analysis and Calculation of Experimental Data
4 Notes
References
Chapter 9: Single-Molecule Nanomechanical Genotyping with DNA Origami-Based Shape IDs
1 Introduction
2 Materials
2.1 Preparation of DNA, Origami-Based Shape IDs
2.2 Labeling of ssDNA Template with Origami-Based Shape IDs
2.3 Agarose Gel Electrophoresis and DNA Extraction
2.4 AFM Imaging
3 Methods
3.1 Preparation of DNA Stock Solutions
3.2 Preparation of DNA Origami Shape IDs
3.3 Purification of DNA Origami Shape IDs
3.4 Preparation of DNA Origami Shape IDs Decorated with STV (Optional)
3.5 AFM Imaging of DNA Origami Shape IDs (With and Without STV)
3.6 Direct Haplotyping of Genomic DNA by DNA Origami Shape IDs
4 Notes
References
Chapter 10: Using Single-Molecule FRET to Evaluate DNA Nanodevices at Work
1 Introduction
2 Materials
2.1 Cleaning of Quartz Microscope Slides
2.2 Functionalization of Quartz Slide and Making Microfluidic Channel
2.3 Buffer Preparation (See Notes 1 and 2)
2.4 Oxygen Scavenger System for smFRET Assay
2.5 Prism-Type Total Internal Reflection Fluorescence Microscopy
3 Methods
3.1 Cleaning of Quartz Microscope Slides
3.2 Surface Functionalization of Quartz Microscope Slides
3.3 Assembling Microfluidic Sample Cells
3.4 Preparation of an Oxygen Scavenging System (OSS)
3.5 Surface Immobilization of Single-DNA Nanodevices and smFRET Data Acquisition
3.6 smFRET Data Analysis
4 Conclusions and Future Outlook
5 Notes
References
Part IV: Applications of DNA and RNA Origami
Chapter 11: Parallel Functionalization of DNA Origami
1 Introduction
2 Materials
2.1 Designing Modular DNA Origami
2.2 Pipetting of Modular Staple Strand Pools
2.3 Copper(I)-Catalyzed Alkyne-Azide Cycloaddition (CuAAC)
2.4 TdT 3′-End Labeling of Selected Staple Strands
2.5 Assembly of DNA Origami with TdT-Functionalized Staple Strands and Gel Electrophoresis
2.6 Characterization of Labeled DNA Origami
3 Methods
3.1 Designing Modular DNA Origami
3.2 Pipetting of Modular Staple Strand Pools
3.3 Synthesis of Modified Nucleotide Triphosphates
3.3.1 Synthesizing a dUTP with a 5 kDa PEG Polymer on the Base
3.3.2 Synthesizing a ddUTP with a Streptavidin (STV) Protein on the Base
3.3.3 Synthesizing a dUTP with a Benzyl on the C5 Position on the Base
3.3.4 Common RP-HPLC Purification Approach
3.4 TdT 3′-End Labeling of Selected Staple Strands (See Notes 13 and 14)
3.4.1 PAGE Characterization of TdT-Labeled Oligonucleotides
3.5 Assembly of DNA Origami with TdT-Labeled Staple Strands
3.6 Characterization of DNA Origami
3.6.1 Agarose Characterization of Labeled DNA Origami
3.6.2 AFM Characterization of Planar DNA Origami Structures (See Notes 25 and 26)
3.6.3 TEM Characterization of Three-Dimensional DNA Origami Structures (See Note 29)
4 Notes
References
Chapter 12: Protein Coating of DNA Origami
1 Introduction
2 Materials
2.1 Assembly and Purification of DNA Origami
2.1.1 DNA Origami
2.1.2 Purification of DNA Origami
2.1.3 Agarose Gel Electrophoresis
2.2 Cationic Protein-Dendron Conjugates
2.2.1 Conjugation
2.2.2 Purification of Protein-Dendron Conjugates with Cation Exchange Chromatography
2.3 Protein Coating and Electrophoretic Mobility Shift Assay
3 Methods
3.1 DNA Origami Assembly
3.2 DNA Origami Purification
3.3 Characterization of DNA Origami Folding and Purification Quality with AGE
3.4 Preparation of BSA-G2 Conjugates
3.5 Conjugate Purification with Cation Exchange Chromatography
3.6 Protein Coating of DNA Origami
4 Notes
References
Chapter 13: Cellular Uptake of DNA Origami
1 Introduction
1.1 Uptake of Particles into Cells
1.2 Design Parameters for DNA Origami Compatible for Cell Uptake
1.2.1 Endotoxin Free Scaffold and Buffers
1.2.2 Fluorescent Labeling
1.2.3 Stability
1.2.4 Uptake Efficiency
1.2.5 Quantity and Purification
2 Materials
2.1 DNA Folding and Protection
2.2 Cell Culture
2.3 DNA Origami Uptake
3 Methods
3.1 Preparation of Cell-Compatible DNA Origami
3.1.1 Endotoxin Free Scaffold (Adapted from)
3.1.2 Folding of the DNA Origami with Sterile Staples and Buffers
3.1.3 Purification and Concentration
3.1.4 Calibration of Fluorescence (Optional, only if Multiple Structures Are to Be Compared)
3.1.5 Coating of DNA Origami for Stabilization
3.2 Cell Culture
3.2.1 Maintenance of Cells
3.2.2 Counting of Cells
3.2.3 Plating Cells of DNA Origami Uptake Assays
3.3 Incubation with DNA Origami
3.3.1 Dilution in Cell Culture Buffer
3.3.2 Addition of DNA Origami to Cells
3.4 Uptake Analysis
3.4.1 Confocal Microscopy
3.4.2 Flow Cytometry
4 Notes
References
Chapter 14: Binding and Characterization of DNA Origami Nanostructures on Lipid Membranes
1 Introduction
2 Materials
2.1 Preparation of Giant Unilamellar Vesicles (GUV)
2.2 Preparation of Multilamellar Vesicles (MLV)
2.3 Preparation of Large Unilamellar Vesicles (LUV)
2.4 Preparation of Supported Lipid Bilayers (SLB)
2.4.1 Specific for Deposition on a Glass Substrate
2.4.2 Specific for Deposition on Top of Freshly Cleaved Mica
2.5 Preparation of Lipid Monolayers
2.6 DNA Origami Folding and Purification
2.7 Fluorescence Confocal Imaging and Correlation Spectroscopy (FCS)
2.8 Negative-Stain Transmission Electron Microscopy (TEM) Imaging
2.9 Atomic Force Microscopy (AFM) Imaging
3 Methods
3.1 Preparation of GUVs by Electroformation (See Note 6)
3.2 Preparation of MLVs
3.3 Preparation of LUVs by Extrusion
3.4 Preparation of SLBs by Vesicle Fusion
3.4.1 Prepare Small Unilamellar vesicles (SUVs) from MLVs (See Subheading 3.2)
3.4.2 Prepare Hydrophilic Substrate of Choice (See Note 9)
3.4.3 Form SLB Via Deposition of SUVs on Top of Hydrophilic Substrate
3.5 Preparation of Lipid Monolayers
3.6 Folding, Purification, and Quantification of DNA Origami Nanostructures
3.6.1 DNA Origami Folding
3.6.2 DNA Origami Purification
3.7 Characterization of DNA Origami Binding to GUVs under Confocal Microscopy
3.8 Characterization of DNA Origami Binding to SLBs under Confocal Microscopy
3.9 Characterization of DNA Origami Binding to SLBs under AFM
3.10 Characterization of DNA Origami Binding to Lipid Monolayers under Confocal Microscopy
3.11 Characterization of DNA Origami Binding to LUVs by Negative-Stain TEM
3.12 Characterization of Diffusion of DNA Origami on Membranes Using FCS
4 Notes
References
Chapter 15: Electrical Actuation of DNA-Based Nanomechanical Systems
1 Introduction
2 Reagents and Materials
2.1 Microscope Setup (See Fig. 2)
2.2 Setup for Binary Electrical Switching
2.3 Preparation of Glass Slides
2.4 Preparation of Origami Structures and Actuation
3 Methods
3.1 Preparation of Origami Structures
3.2 Preparation of Glass Slides
3.3 A Simple Setup for Binary Electrical Switching (see Fig. 2)
4 Design Considerations for Electrically Driven DNA Nanostructures
5 Observation and Microscopy
6 A Sample Chamber for 2D Rotational Actuation
7 Custom Electrode Plugs for 2D Rotational Actuation
8 High Voltage Amplifier for Computer-Controlled Actuation (See Note 7)
9 Overall Experimental Procedure
9.1 Setting Up an Experiment
9.2 Tracking of Actuated Arms and Data Processing
10 Performance and Application Potential
11 Notes
References
Chapter 16: Enzyme Cascade Reactions on DNA Origami Scaffold
1 Introduction
2 Materials
2.1 Buffers and Reagents
2.2 Equipment
3 Methods
3.1 Preparation of DNA Origami with the Modification Sites for Protein Assembly
3.1.1 Design of DNA Origami with Modification Sites for Protein Assembly
3.1.2 Preparation of Tag-Substrate Modified ODNs (See Fig. 2b-d)
3.1.3 Preparation and Purification of DNA Origami Scaffold with the Modification Sites (See Fig. 3a, b)
3.2 Design and Preparation of the DNA Binding Adaptor-Fused POI
3.3 Preparation and Characterization of Adaptor-Fused POI Assembled DNA Origami Scaffold
3.3.1 Preparation of POI Assembled DNA Origami Scaffold
3.3.2 Characterization of the POI Assembled DNA Origami Scaffold
3.4 Enzyme Cascade Reactions on the DNA Origami Scaffold
3.4.1 Enzyme Assay for a Single Enzyme
3.4.2 Two Enzyme Cascade Reaction on DNA Origami Scaffold
3.4.3 Three Enzyme Cascade Reaction on DNA Origami Scaffold
4 Notes
References
Chapter 17: Aptamers as Functional Modules for DNA Nanostructures
1 Introduction
2 Materials
2.1 Buffers
2.2 DNA Strands, Proteins, and Purification Columns
2.3 Droplet PCR, Picoinjection, and Transcription
2.4 Microfluidic Devices
2.5 Circular Dichroism
2.6 Synthesis and Quality Control of Aptamer Beads
2.7 On-Bead Binding Assay
2.8 G-Quadruplex Peroxidase Assay
2.9 Aptamer-Tethered Enzyme Capture (APTEC) Assay
2.10 Nanostructure Characterization by Transmission Electron Microscopy (TEM)
2.11 Nanostructure Imaging by Atomic Force Microscopy (AFM)
3 Methods
3.1 DNA Nanostructure Assembly
3.2 Aptamer Evolution Using Microfluidic SELEX
3.2.1 Aptamer Library Preparation
3.2.2 Fabrication of Microfluidic Devices
3.2.3 Droplet PCR
3.2.4 Picoinjection and In Vitro Transcription
3.2.5 Droplet Sorting
3.3 Aptamer Refolding
3.4 Aptamer Characterization by Circular Dichroism
3.5 On-Bead Aptamer Affinity Assay
3.5.1 Synthesis and Quality Control of Aptamer Beads
3.5.2 Synthesis of Forward Priming Beads
3.5.3 Concentration Measurement
3.5.4 On-Bead Polymerase Chain Reaction (bPCR) to Generate Aptamer Particles
3.5.5 Aptamer Bead Quality Control by Flow Cytometry
3.5.6 On-Bead Aptamer Binding Assay
3.6 Determine Optimal Aptamer-Duplex Competition
3.7 G-Quadruplex Peroxidase Assay
3.8 Quantification of Conformational Change using Fluorescence Resonance Energy Transfer (FRET)
3.8.1 Standard Curve Preparation
3.8.2 PfLDH-Mediated Opening of DNA Origami Box Assessed by FRET Assay
3.9 Aptamer-Tethered Enzyme Capture (APTEC) Assay
3.9.1 Functionalization of 96-Well Plates
3.9.2 APTEC Assay
3.9.3 APTEC Assay in Whole Rat Blood
3.10 Nanostructure Imaging by Transmission Electron Microscopy (TEM)
3.10.1 Negative Staining (see Note 42)
3.10.2 Observation Under an Electron Microscope
3.11 Nanostructure imaging by Atomic Force Microscopy (AFM)
3.11.1 AFM Air Mode Protocol
3.11.2 AFM Liquid Mode Protocol (Should Be Conducted Swiftly to Minimize Evaporation)
4 Notes
References
Chapter 18: Production and Testing of RNA Origami Anticoagulants
1 Introduction
2 Materials
2.1 DNA Template Amplification and Purification
2.2 Agarose Gel Electrophoresis
2.3 RNA Origami Production Using In Vitro Transcription and Purification
2.4 Components for Native and Denaturing Acrylamide Gel Electrophoresis
2.5 Folding RNA Origami by Heat-Annealing Procedure
2.6 Anticoagulation Test: aPTT Assay
3 Methods
3.1 DNA Template Production
3.2 Characterization of the Amplified DNA Template Using Agarose Gel Electrophoresis
3.3 In Vitro Transcription of RNA Origami
3.3.1 Transcription of Native RNA Origami
3.3.2 Transcription of 2′-Fluoro Modified RNA Origami
3.4 Characterization of RNA Origami
3.5 Folding RNA Origami
3.6 Anticoagulation Activity Test by aPTT Assay
4 Notes
References
Index