RNA Nanostructures: Design, Characterization, and Applications (Methods in Molecular Biology, 2709)

✍ Scribed by Kirill A. Afonin (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 331
• Edition: 1st ed. 2023
• Category: Library

✦ Synopsis

This volume details protocols for computer-assisted design and experimental characterization of RNA nanostructures. Chapters guide readers through RNA nanotechnology, design and characterization of RNA nanostructures, assessment of immunology of nanomaterials, biosensing and drug delivery, various biomedical applications, and delivery approaches for therapeutic RNA nanoparticles. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

  Authoritative and cutting-edge, RNA Nanostructures: Design, Characterization, and Applications aims to address essential topics and concerns in the growing field of RNA nanotechnology.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Computational Design and In Silico Studies of RNA Nanostructures
Chapter 1: Molecular Dynamics Simulations of RNA Motifs to Guide the Architectural Parameters and Design Principles of RNA Nan...
1 Introduction
2 Materials
2.1 AMBER
2.2 CHARMM and NAMD
2.3 VMD (Visual Molecular Dynamics)
2.4 Discovery Studio Visualizer (DSV)
3 Methods
3.1 MD Simulation of a Linear RNA Motif
3.1.1 Preparation of Initial Structure and Topology Using AMBER FF
3.1.2 Initial Minimization and Equilibration
3.1.3 Constrained MD Simulation
3.1.4 Final Equilibration and Product MD Simulations
3.2 MD Simulation of a Bent RNA Motif and RNA Nanoring
3.3 Data Analysis Using cpptraj and VMD
3.3.1 cpptraj
3.3.2 VMD
4 Notes
References
Chapter 2: Computer-Assisted Design and Characterization of RNA Nanostructures
1 Introduction
2 Materials
2.1 Initial RNA 3D Structure Prediction
2.2 Force Fields
2.3 Software
3 Methods
3.1 Explicit Solvent Simulations of NANPs
3.1.1 Preparation of the System
3.1.2 Initial Minimization
3.1.3 Equilibration
3.1.4 Production Run
3.2 Implicit Solvent Simulations of NANPs
3.2.1 Preparation of the System
3.2.2 Initial Minimization
3.2.3 Equilibration
3.2.4 Production Run
3.3 Characterization
3.3.1 Potential Energy
3.3.2 Radius of Gyration and RMSD
4 Notes
References
Chapter 3: Combining Experimental Restraints and RNA 3D Structure Prediction in RNA Nanotechnology
1 Introduction
1.1 Utilize NMR Restraints for RNA 3D Structure Prediction
1.2 Better Utilize Distance Restraints for RNA 3D Structure Prediction
2 Materials
3 Methods
3.1 Coarse-Grained Molecular Dynamics for RNA: iFoldRNA
3.2 All-Atom RNA Modeling
3.3 Utilize NMR Restraints for RNA 3D Structure Prediction: iFoldNMR
3.4 Better Utilize Distance Restraints for RNA 3D Structure Prediction: DVASS
4 Notes
References
Chapter 4: Structural Characterization of Nucleic Acid Nanoparticles Using SAXS and SAXS-Driven MD
1 Introduction
2 Materials
2.1 SAXS Sample Preparation and Data Collection
2.2 SAXS-Driven Molecular Dynamics Simulations
3 Methods
3.1 Preparation of Samples for Measurement at Beamline
3.2 For SEC-SAXS Measurement Preparation Skip to Subheading 3.3. For HT-SAXS:
3.3 Preparing SEC-SAXS Samples
3.4 SAXS/WAXS Data Processing for High-Throughput Static Experiments
3.5 SAXS/WAXS Data Processing for Size-Exclusion Chromatography Coupled with SAXS (SEC-SAXS)
3.6 SAXS-Driven Molecular Dynamics
3.7 Free MD Calculation for Solvent-Water and Ions
3.8 SAXS-Driven MD
3.9 Output Files and Analysis
3.10 Rerun SAXS-MD Module
4 Notes
References
Part II: Production and Storage of Functional RNA Nanostructures
Chapter 5: Metalated Nucleic Acid Nanostructures
1 Introduction
2 Materials
2.1 Buffers
2.2 Nucleic Acid Stock Solutions
2.3 Silver Nitrate Stock Solutions
2.4 Native Polyacrylamide Gel
3 Methods
3.1 Nucleic Acid Samples
3.2 Native Gel Electrophoresis
3.3 Atomic Force Microscopy (AFM)
4 Notes
References
Chapter 6: Bioconjugation of Functionalized Oligodeoxynucleotides with Fluorescence Reporters for Nanoparticle Assembly
1 Introduction
2 Materials
2.1 Chemicals
2.2 Instrumentation
3 Methods
3.1 Conjugation of ODN Alkyne with 3-Azido-7-Hydroxycoumarin
3.2 Conjugation of ODN Azide with Cy3 Alkyne
3.3 Confirmation of Clicked Products on Denaturing Polyacrylamide Gel Electrophoresis (PAGE)
3.4 Purification of the DNA Conjugates Using Molecular Weight Cutoff Membrane (MWCO) and Analysis by Fluorescence Spectroscopy
3.4.1 Purification of Clicked Products
3.4.2 Fluorescence Assay of ODN Conjugates Before and After Purification
3.5 DNA Nanoparticle Self-Assembly with the ODN-Coumarin and ODN-Cy3 Conjugates
4 Notes
References
Chapter 7: Light-Assisted Drying for the Thermal Stabilization of Nucleic Acid Nanoparticles and Other Biologics
1 Introduction
2 Materials
2.1 LAD Processing and Storage
2.2 Polarized Light Imaging
3 Methods
3.1 LAD Processing and Sample Storage
3.2 Calculation of Sample End Moisture Content
3.3 Determination of Appropriate Processing Time
3.4 Polarized Light Imaging (PLI)
4 Notes
References
Chapter 8: Preparation of Nucleic Acid Aptamer Functionalized Silver/Gold Nanoparticle Conjugates Using Thiol-Substituted Olig...
1 Introduction
2 Materials
2.1 Deprotection of Aptamers
2.2 Generating Gold Nanoparticle-Aptamer Conjugates with 1:1 to 1:100 Nanoparticle-Aptamer Ratio
2.3 Generating Gold Nanoparticle-Aptamer Conjugates with Many Aptamers per Particle
2.4 Generating Silver Nanoparticle-Aptamer Conjugates
2.5 Confirming attachment of aptamers to nanoparticles
2.5.1 Agarose Gel Electrophoresis
2.5.2 Denaturing Gel Electrophoresis
2.5.3 Determining Aptamer-Nanoparticle Ratios
2.6 Facile Synthesis of AuNPs 15-20 nm in Size
3 Methods
3.1 Deprotection of Aptamers.
3.2 Preparation of AuNP-RNA Conjugates
3.2.1 Preparation of Conjugates with Low AuNP-Aptamer Ratios (1:1, 1:10, or 1:100)
3.2.2 Preparation of Conjugates with High Aptamer-AuNP Ratios (Coat the Entire NP Surface)
3.3 Purification of AuNP-Aptamer Conjugates
3.4 Preparation of AgNP-Aptamer Conjugates
3.5 Purification of AgNP-Aptamer Conjugates
3.6 Confirmation of Aptamer Attachment
3.6.1 Agarose Gel Electrophoresis (Nondenaturing)
3.6.2 UV-VIS Spectrometry
3.6.3 Denaturing Polyacrylamide Gel Electrophoresis (Urea Gel)
3.6.4 Dynamic Light Scattering
3.7 Determining NP-Aptamer Ratios
3.8 Facile Synthesis of AuNPs
4 Notes
References
Part III: Characterization of RNA Nanostructures
Chapter 9: Thermodynamic Characterization of Nucleic Acid Nanoparticles Hybridization by UV Melting
1 Introduction
2 Materials
2.1 Chemicals
2.2 UV-Visible Spectrophotometer
2.3 Buffering System
3 Methods
3.1 Nucleic Acid Sample Preparation and Experimental Design
3.2 Data Analysis
4 Notes
References
Chapter 10: Structural Characterization of DNA-Templated Silver Nanoclusters by Energy Dispersive Spectroscopy
1 Introduction
1.1 Identification and Quantification of Elements
1.2 Importance of Beam Alignment
1.3 Astigmatism Correction
1.4 Aperture Alignment
1.5 Choosing Ideal Instrument Settings for EDS
1.6 Accelerating Voltage
1.7 Working Distance
1.8 Dead Time and Process Time
1.9 Spot Size
1.10 Aperture
1.11 Beam Damage and Charging Effects
1.12 Sample Preparation
1.13 Sample Substrate Choice
1.14 Elements of Interest
2 Materials
2.1 Sample Wash
2.2 Substrate
2.3 Sample Drying
2.4 Sample Transportation
2.5 Sample Mounting
2.6 SEM/EDS
3 Methods
3.1 Sample Preparation and Deposition on Silicon Substrate
3.2 SEM/EDS Analysis
4 Notes
References
Chapter 11: Small Volume Microrheology to Evaluate Viscoelastic Properties of Nucleic Acid-Based Supra-Assemblies
1 Introduction
2 Materials
3 Methods
3.1 Supra-Assembly Synthesis
3.2 Particle Tracking Setup and Its Calibration
3.3 Bead Suspension Preparation and Video Acquisition
3.3.1 Glycerol/Water Mixtures
3.4 Particle Tracking and Data Analysis
4 Notes
References
Chapter 12: Characterization of RNA Nanoparticles and Their Dynamic Properties Using Atomic Force Microscopy
1 Introduction
2 Materials and Equipment
2.1 RNA Nanoparticle Preparation
2.2 Mica Preparation and Sample Deposition
2.3 AFM Imaging and Data Analysis
3 Methods
3.1 RNA Nanoparticle Preparation
3.2 Mica Surface Modification with 1-(3-Aminopropyl)Silatrane (APS)
3.3 Deposition of RNA Nanoparticles on APS-Modified Mica Surface
3.4 Atomic Force Microscopy Imaging
3.4.1 Brief Overview
3.4.2 Atomic Force Microscopy Imaging
3.4.3 AFM Image Analysis to Evaluate Dynamicity of Nanoring Structures
4 Notes
References
Part IV: Intracellular Delivery and Immunorecognition of RNA Nanostructures
Chapter 13: Synthesis of Mesoporous Silica Nanoparticles for the Delivery of Nucleic Acid Nanostructures
1 Introduction
2 Materials
2.1 CTAB Micelle Formation
2.2 Surface Modification of the Material
2.3 Surfactant Removal
2.4 Post-grafting and Primary Amine Quantification
2.5 Nucleic Acid NP Formation
3 Methods
3.1 Synthesis of Negatively Charged Mesoporous Silica Nanoparticles (MSNPs)
3.2 Synthesis of PEG-PEI-Modified MSNPs
3.3 Kaiser´s Assay
3.4 In Vitro Run-Off T7 Transcription of ssRNAs
3.5 Synthesis of NANPs
3.6 Complexation of NA-MS-NPs
4 Notes
References
Chapter 14: Assessment of Intracellular Compartmentalization of RNA Nanostructures
1 Introduction
2 Materials
2.1 Statistical Analysis
3 Methods
3.1 Statistical Analysis
4 Notes
References
Chapter 15: Discriminating Immunorecognition Pathways Activated by RNA Nanostructures
1 Introduction
2 Materials and Equipment
2.1 NANPs Synthesis
2.2 UV-Melt
2.3 Dynamic Light Scattering (DLS)
2.4 Fetal Bovine Serum Stability Assay
2.5 Source and Propagation of Cell Lines
2.6 Reporter Cell Lines
2.7 Transfection of Microglia
2.8 siRNA Knockdown
2.9 Quantification of Cytokines in Cell Supernatants
2.10 Immunoblot Analysis
3 Methods
3.1 Synthesis of NANPs
3.2 UV-Melt Experiments
3.3 DLS
3.4 FBS Stability Assay
3.5 Cell Line Propagation
3.6 Reporter Cell Lines
3.7 Microglia Transfection
3.8 siRNA Knockdown
3.9 Quantification of Cytokines in Cell Supernatants
3.10 Immunoblot Analysis
4 Notes
References
Chapter 16: Detection of Nanoparticles´ Ability to Stimulate Toll-Like Receptors Using HEK-Blue Reporter Cell Lines
1 Introduction
2 Materials
3 Methods
3.1 Cell and Reagent Preparation
3.2 Experimental Procedure
3.3 Calculations and Criteria for Assay Acceptance
4 Notes
References
Chapter 17: Characterization of PAMAM Dendrimers for the Delivery of Nucleic Acid Nanoparticles
1 Introduction
2 Materials and Equipment
2.1 Nucleic Acid Nanoparticle (NANP) Preparation
2.2 Determination of N/P Ratio Using Binding Assays
2.3 Nuclease Protection Assay
2.4 Competitive Binding Assay
3 Methods
3.1 Nanoparticle Preparation
3.2 Binding Assays to Experimentally Determine and Confirm N/P Ratios
3.3 Nuclease Protection Assay
3.4 Competitive Binding Assay
4 Notes
References
Part V: RNA and DNA Nanostructures Designed for Biomedical Applications
Chapter 18: Reverse Transfection of Functional RNA Rings into Cancer Cells Followed by in Vitro Irradiation
1 Introduction
2 Materials
2.1 Cancer Mammalian Cell Culture
2.2 Synthesis of RNA Rings
2.3 Reverse Transfection
2.4 Irradiation
3 Methods
3.1 Cancer Mammalian Cell Culture
3.2 Synthesis of RNA Rings
3.3 Reverse Transfection of RNA Rings into Mammalian Cancer Cells
3.4 Irradiation of Mammalian Cancer Cells Transfected with RNA Rings
4 Notes
References
Chapter 19: Aptamer Conjugated RNA/DNA Hybrid Nanostructures Designed for Efficient Regulation of Blood Coagulation
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Study Samples
3.2 Preparation of Test and Normal and Abnormal Control Plasmas
3.2.1 Blood Sample Preparation and General Testing Guidelines
3.2.2 Test Plasma (With Aptamer-NANPs)
3.2.3 Normal and Abnormal Control Plasmas
3.2.4 Neoplastin, PTT-A Reagent, and Thrombin Preparation (Used to Initiate Plasma Coagulation)
3.3 Plasma Coagulation Assay Procedure
3.4 Calculations and Data Interpretation
4 Notes
References
Chapter 20: Detection of Multiplex NASBA RNA Products Using Colorimetric Split G Quadruplex Probes
1 Introduction
2 Materials
2.1 General Supplies and Equipment
2.2 NASBA Amplification Reaction
2.3 Visual Detection of Amplicons
3 Methods
3.1 Sensor Design
3.2 NASBA Amplification Reactions
3.3 Visual Detection of the NASBA Amplicons
4 Notes
References
Chapter 21: Synthesis of DNA-Templated Silver Nanoclusters and the Characterization of Their Optical Properties and Biological...
1 Introduction
1.1 Structure and Function of DNA-AgNCs
1.2 Demand for New Antibacterial Treatments
1.3 Characterizing the Antibacterial Effectiveness and Testing Biocompatibility of Mammalian Cells
2 Materials
2.1 Synthesis of DNA-AgNCs
2.2 Fluorescence Experiment
2.3 Bacterial Growth Assays
2.4 Mammalian Cell Viability Assays
3 Methods
3.1 Synthesis of DNA-AgNCs
3.2 Fluorescence Experiment
3.3 Bacterial Growth Assays (Fig. 2)
3.4 Mammalian Cell Viability Assays (Fig. 3)
4 Notes
References
Chapter 22: Dynamic Nanostructures for Conditional Activation and Deactivation of Biological Pathways
1 Introduction
2 Materials
2.1 Synthesis and Characterization of Hybrid RNA/DNA Fibers
2.2 Assessment of Biological Activity of NF-κB Decoy Fibers
2.2.1 Primary Human Peripheral Blood Mononuclear Cells (PBMCs) for Analysis of Interferon and Cytokine Secretion (to Assess An...
2.2.2 Reporter Cell-Based Assay (to Assess Anti-inflammatory Potential)
2.2.3 Immunofluorescence Analysis for Detection of NF-κB in Cancer Cell Line (to Assess Biological Activity)
2.3 Statistical Analysis
3 Methods
3.1 Synthesis and Physicochemical Characterization of Fibers
3.2 Assessment of NF-κB Biological Activity in Cell Models
3.2.1 Primary Human Peripheral Blood Mononuclear Cells (PBMCs) and Whole-Blood Culture for Analysis of Cytokine Secretion (See...
3.2.2 Reporter Cell-Based Assay for Assessment of NF-κB-Dependent SEAP (See Note 4)
3.2.3 Immunofluorescence Analysis for Detection of NF-κB in Cancer Cells (See Note 5)
3.3 Statistical Analysis
4 Notes
References
Chapter 23: Anticoagulant Activity of Nucleic Acid Nanoparticles (NANPs) Assessed by Thrombin Generation Dynamics on a Fully A...
1 Introduction
2 Materials
2.1 Anticoagulant and Antidote Fiber NANP Preparation
2.2 Blood Collection and Plasma Preparation
2.3 Calibration
2.4 Quality Controls
2.5 End-of-Day Procedure
3 Methods
3.1 Anticoagulant and Antidote Fiber Preparation
3.2 Blood Collection and Plasma Preparation
3.3 Turning the Analyzer on and Priming the System at Startup
3.4 Calibration
3.5 Quality Controls
3.6 Samples
3.7 End-of-Day Procedure
4 Notes
References
Index