Therapeutic RNA Nanotechnology: Immunomodulation and Dynamicity

Cover
Half Title
Title Page
Copyright Page
Dedication
Table of Contents
Preface
Part I: Nanodesign
Chapter 1: Generating New Specific RNA Interaction Interfaces Using C-Loops
1.1: Introduction
1.2: Materials and Methods
1.2.1: Design of the C-Loop-Containing Tectonics
1.2.2: RNA Preparation
1.2.3: pCp Labeling of RNA Molecules
1.2.4: Assembly Experiments
1.2.5: Dissociation Constants (Kd) Determination
1.2.6: Lead (Pb2+)-Induced Cleavage
1.3: Results
1.3.1: Design and Nomenclature of C-Loop-Containing Tecto-RNA Molecules
1.3.2: Assembly Experiments and Dissociation Constants (Kd’s)
1.3.3: Competition Experiments
1.3.4: Monitoring C-Loop Formation by Pb(II)-Induced Cleavage
1.4: Discussion
1.5: Conclusion
Chapter 2: Specific RNA Self-Assembly with Minimal Paranemic Motifs
2.1: Introduction
2.2: Materials and Methods
2.2.1: Sequence Design
2.2.2: RNA Preparation
2.2.3: Radio-Labeling of RNA Molecules
2.2.4: Assembly Experiments
2.2.5: Determination of Dissociation Constants (Kd)
2.2.6: Lead (Pb2+)-Induced Cleavage
2.3: Results
2.3.1: Designs of RNA Molecules for Paranemic Assembly
2.3.2: Paranemic RNA Self-Assembly
2.3.3: Using Pb(II) to Probe the Formation of Paranemic Complexes
2.3.4: Kinetic Exchange
2.3.5: Sequence Specificity
2.3.6: Determination of Dissociation Constant for 3HT Complexes
2.4: Discussion
2.5: Conclusion
Chapter 3: In vitro Assembly of Cubic RNA-Based Scaffolds Designed in silico
3.1: Introduction
3.2: Rational Design of a Nanocube
3.3: Nanocube Self-Assembly
3.4: Comparison of RNA, DNA and RNA/DNA Nanocubes
3.5: Structural Characterization by DLS and Cryo-EM
3.6: RNA Nanocube Functionalization
3.7: Conclusion
3.8: Methods
3.8.1: RNA Preparation
3.8.2: pCp Labelling of RNA Molecules
3.8.3: ATP Labelling of DNA
3.8.4: Self-Assembly of RNA and DNA Cubes
3.8.5: Non-denaturing PAGE, TGGE Experiments and Kd Measurements
3.8.6: Assembly of RNA Cubes during Transcription
3.8.7: Dynamic Light Scattering
3.8.8: Cryo-EM Imaging
3.8.9: Fluorescence Experiments
Chapter 4: Self-Assembling RNA Nanorings Based on RNAI/II Inverse Kissing Complexes
4.1: Introduction
4.2: Results and Discussion
4.2.1: Nanoring Design and Self-Assembly
4.2.2: Structural Characterization of Nanoring Assembly
4.2.3: Exploring Structural Flexibility versus Structural Rigidity on Nanoring Assembly
4.2.4: Design and Characterization of Programmable Nanorings
4.2.5: Nanorings with Increased Resistance toward Ribonuclease Degradation in Blood Serum
4.2.6: Nanoring Functionalization with siRNAs
4.3: Conclusion
4.4: Materials and Methods
4.4.1: RNA Structural and Sequence Design
4.4.2: RNA Synthesis
4.4.3: RNA Assembly, Native PAGE, and TGGE Experiments
4.4.4: AFM Characterization
4.4.5: Specificity Study and Determination of Equilibrium Constant of Dissociation (Kd)
4.4.6: Human Blood Serum Degradation Studies
4.4.7: Recombinant Human Dicer Assays
Chapter 5: Multistrand RNA Secondary Structure Prediction and Nanostructure Design Including Pseudoknots
5.1: Introduction
5.2: Results
5.2.1: Quality of Structure Prediction
5.2.2: Web Server
5.3: Discussion
5.4: Materials and Methods
5.4.1: RNA Structure Data Sets
5.4.2: Multistrand Structure Prediction Including Pseudoknots
5.4.2.1: Scoring of RNA structures
5.4.2.2: RNA folding algorithm
5.4.3: RNA Sequence Design Algorithm
5.4.3.1: Secondary structure similarity component
5.4.3.2: Sequence design rule component
5.4.3.3: Sequence optimization algorithm
5.4.3.4: Secondary structure prediction programs used for comparison
5.4.3.5: RNAcofold
5.4.3.6: Pairfold
5.5: Experimental Procedures
5.5.1: RNA Preparation
5.5.2: Co-transcriptional α[P32]-ATP Body Labeling of RNA Molecules
5.5.3: Nondenaturing PAGE Experiments
5.5.4: RNA Sequences Used in This Example
5.5.5: Three-Way Junction Motif
Chapter 6: Computational and Experimental Characterization
of RNA Cubic Nanoscaffolds
6.1: Introduction
6.2: The Use of Coarse-Grained Structure Dynamics Characterization with ANM
6.3: Computational and Experimental Nanocube Design and Assembly
6.3.1: Computational Generation of RNA Cube Models Predicts Best Design Choices
6.3.2: Native PAGE Experiments Justify Design Choices
6.4: Experimental and Computational Determination of Nanocubes’ Sizes
6.4.1: Dynamic Light Scattering (DLS) Experiments
6.4.2: Applying ANM to Predict the Size Limits of Nanocube Motions
6.5: Melting Properties of the Cubes
6.5.1: Thermal Gradient Gel Electrophoresis (TGGE) Experiments
6.5.2: Calculating the Relative Strain and Flexibility of Cubes’ Structural Components
6.6: Conclusion
6.7: Experimental and Computational Procedures
6.7.1: Experimental Methods
6.7.1.1: RNA preparation
6.7.1.2: [32P]Cp labeling of RNA molecules
6.7.1.3: Non-denaturing PAGE experiments
6.7.1.4: Thermal gradient gel electrophoresis (TGGE) experiments
6.7.1.5: Dynamic light scattering
6.7.2: Computational Methods
6.7.2.1: Modeling
6.7.2.2: Anisotropic network model
6.7.2.3: Energy minimization
Chapter 7: In silico Design and Enzymatic Synthesis of Functional RNA Nanoparticles
7.1: Introduction
7.2: Nanostructure Modeling Strategies
7.3: Nanostructure Characterization
7.4: Multistrand Secondary Structure Prediction and Sequence Design
7.5: Enzymatic Production of RNA Nanoparticles
7.6: In vivo Delivery of RNA Nanoparticles
7.7: Conclusion
Chapter 8: Ring Catalog: A Resource for Designing Self-Assembling RNA Nanostructures
8.1: Introduction
8.2: Methods and Materials
8.2.1: Generation of the Ring Catalog
8.2.1.1: Choice of motifs
8.2.1.2: Generation of Ring structures
8.2.1.3: Topology classification
8.2.2: Post-processing of Ring-Structures
8.2.3: Motif Used for Experimentally Verified Structure
8.2.4: Experimental Assembly of the RNA Nanoconstruct
8.3: Results
8.3.1: Ring Catalog
8.3.2: Post-processing of Ring Structures
8.3.3: Results of Experimental Assemblies
8.4: Discussion
8.5: Conclusion
Chapter 9: Programmable RNA Microstructures for Coordinated Delivery of siRNAs
9.1: Introduction
9.2: Materials and methods
9.2.1: Oligonucleotides
9.2.2: RNA Extraction
9.2.3: RNA Construct Purification
9.2.4: Assembly Methods
9.2.5: Atomic Force Microscopy (AFM)
9.2.6: Denaturing PAGE
9.2.7: Non-denaturing PAGE
9.2.8: Dynamic Light Scattering (DLS) Experiments
9.2.9: UV-Melting Experiments
9.2.10: Human Blood Serum Stability Assays
9.2.11: Transfection Experiments
9.2.12: Flow Cytometry
9.2.13: Microscopy
9.2.14: Viability Assays
9.3: Results
9.3.1: Design of Functional Tiles
9.3.2: Tile and Lattice Assembly and Characterization
9.3.3: Transfection of Tiles and Lattices
9.3.4: Functional Tiles and Lattices Successfully Silence Target Genes
9.4: Discussion
Chapter 10: Versatile RNA Tetra-U Helix Linking Motif as a
Toolkit for Nucleic Acid Nanotechnology
10.1: Introduction
10.2: Methods
10.2.1: Triangular Nanoscaffolds Self-Assembly
10.2.2: Electrophoretic Mobility Shift Assay
10.2.3: UV-Melting Experiments
10.2.4: Fetal Bovine Serum Degradation Assay
10.2.5: AFM Imaging and Sample Preparation
10.2.6: Transfection of Human Cell Lines
10.2.7: Fluorescent Microscopy and Flow Cytometry
10.2.8: Immunotoxicity Experiments
10.3: Results
10.3.1: Rationale Behind Application and Design of Artificial RNA 4: Us Helical Linking Motif
10.3.2: Nucleic Acid Triangular Nanoscaffolds Assembly and Characterization
10.3.3: Thermal Stabilities of Triangular Nanoscaffolds
10.3.4: Chemical Stabilities of Triangular Nanoscaffolds
10.3.5: Immunostimulatory Activities of Triangular Nanoscaffolds
10.3.6: RNA/DNA-Center Hybrid
Nanoscaffold Carrying siRNA
Demonstrate Efficient Gene Silencing Properties
10.4: Discussion
Part II: Functional RNA Nanoparticles and Their Delivery
Chapter 11: Design and self-Assembly of siRNA-Functionalized RNA Nanoparticles for Use in Automated Nanomedicine
11.1: Introduction
11.1.1: Experimental Design
11.1.1.1: Nanodesign strategy 1 (nanoring scaffold)
11.1.1.2: Nanodesign strategy 2 (nanocube scaffold)
11.1.2: RNA Scaffold and siRNA Sequence Design
11.1.3: RNA Preparation
11.1.4: Essential Formulation Quality Control Before Safety Evaluation for Biomedical Applications
11.1.5: Cassette-Based Assembly Protocols for Functionalized NPs
11.1.5.1: One-pot assembly
11.1.5.2: Stepwise assembly
11.1.5.3: Premade siRNA duplex assembly
11.2: Materials
11.3: Procedure
11.4: Anticipated Results
11.4.1: Anticipated Assembly Results
11.4.2: Anticipated Functional Control Results
11.4.3: Anticipated Endotoxin Screening Results
Chapter 12: Co-transcriptional Assembly of Chemically Modified RNA Nanoparticles Functionalized with siRNAs
Chapter 13: Multifunctional RNA Nanoparticles
13.1: Introduction
13.2: Functional Nanoring Assembly and Characterization
13.3: Nanoring-Mediated Gene Silencing and Cell Targeting in vitro
13.4: Functionalization of Nanorings through Toehold Interactions
13.5: Controlled Activation of Intracellular FRET and RNAi by Nanorings with RNA-DNA Hybrids
13.6: Implementation of Functional Nanorings in vivo
13.7: Functional Nanorings against HIV-1
13.8: Methods Summary
13.8.1: RNA Nanoring Sequence Design Assemblies and Native PAGE
13.8.2: Dynamic Light Scattering (DLS) Experiments
13.8.3: Recombinant Human Dicer Assay
13.8.4: Malachite Green (MG) Aptamers Fluorescent Experiments
13.8.5: Cryo-Electron Microscopy (Cryo-EM) Experiments
13.8.6: Cryo-EM Reconstruction
13.8.7: Hexameric Nanoring Models
13.8.8: Fitting Hexameric Nanoring Models to the Cryo-EM Density Map
13.8.9: Transfection Experiments
13.8.10: Microscopy
13.8.11: Endosomal Colocalization Studies
13.8.12: Reassociation of RNA-DNA Hybrids in Cells Assessed through FRET
13.8.13: Flow Cytometry Experiments
13.8.14: Cell Viability Assay
13.8.15: In vivo Silencing Experiments
13.8.16: HIV-1 Inhibition by Functional Nanorings
Chapter 14: Oxime Ether Lipids Containing Hydroxylated Head Groups are More Superior siRNA Delivery Agents Than Their
Nonhydroxylated Counterparts
14.1: Introduction
14.2: Materials and Methods
14.2.1: Materials
14.2.2: Methods
14.2.2.1: Synthesis of oxime ether lipids
14.2.3: Nucleic Acid Duplex Assembly
14.2.4: Preparation of Oxime Ether Liposomes
14.2.5: Formation of Oxime Ether Liposomes/Nucleic Acid Complexes
14.2.6: Size & Zeta-Potential Measurements
14.2.7: Cryo-Electron Microscopy
14.2.8: Fluorescent Anisotropy/Polarization Measurements
14.2.9: Protection of Nucleic Acids
14.2.10: Cell Culture Studies
14.2.11: Uptake & Silencing Measurement by Flow Cytometry
14.2.12: Fluorescent Microscopy
14.2.13: Endosomal Colocalization
14.2.14: Cell Viability Assay
14.3: Results
14.3.1: Hydrodynamic Diameter Analysis
14.3.2: Zeta-Potential Measurements
14.3.3: Morphology
14.3.4: Binding Affinity of Oxime Ether Lipid/Nucleic Acid Complexes
14.3.5: Protection of Nucleic Acids by OEL
14.3.6: Cell Viability
14.3.7: Uptake Efficiency of Nucleic Acids by OELs
14.3.8: Silencing of Green Fluorescent Protein Gene
14.3.9: Endosomal Colocalization
14.4: Discussion
14.5: Conclusion
14.6: Future Perspective
Chapter 15: Bolaamphiphiles as Carriers for siRNA Delivery: From Chemical Syntheses to Practical Applications
15.1: Introduction
15.2: Material and Methods
15.2.1: Syntheses of the Bolaamphiphile Compounds
15.2.2: Nucleic Acid Duplex Assembly
15.2.3: Formation of Bola/Duplex Complexes
15.2.4: Fluorescent Anisotropy/Polarization Measurements
15.2.5: Nuclease Degradation
15.2.6: Cell Culture Studies
15.2.7: Cell Viability Assay
15.2.8: Transfection and Silencing Measurements by Flow Cytometry
15.2.9: Fluorescent Microscopy
15.2.10: Endosomal Colocalization Experiments
15.2.11: Transfection of DS RNAs Designed Against HIV
15.2.12: Dynamic Light Scattering (DLS) Experiments
15.2.13: Cryo-EM Experiments
15.2.14: In silico Studies of Bolas (MD Simulation)
15.3: Results
15.3.1: Syntheses of Bolaamphiphiles
15.3.2: Experimental Characterization of Bolaamphiphiles
15.3.2.1: Fluorescent anisotropy/polarization
15.3.2.2: Size analysis with DLS
15.3.2.3: Cryo-EM imaging
15.3.2.4: Nuclease degradation assays
15.3.2.5: Cellular uptake experiments
15.3.2.6: Endosomal colocalization
15.3.2.7: Gene silencing efficiency
15.3.2.8: Cell viability assay
15.3.2.9: Transfection of DS RNAs designed against HIV
15.3.3: MD Simulations
15.3.3.1: Micelle formation
15.3.3.2: Aggregation of bolas on RNA surfaces
15.4: Discussion
15.5: Conclusion
Chapter 16: Magnetic Nanoparticles Loaded with Functional RNA Nanoparticles
16.1: Introduction
16.2: Experimental
16.2.1: Materials
16.2.2: Iron Oxide Nanoparticle Synthesis
16.2.3: Nanoparticle Aqueous Phase Transfer
16.2.4: Nanoparticle Coating with Polyethyleneimine (PEI)
16.2.5: RNA Nanoparticle Synthesis
16.2.6: Atomic Force Microscopy (AFM)
16.2.7: Dynamic Light Scattering (DLS) and Zeta Potential Measurements
16.2.8: Transmission Electron Microscopy (TEM)
16.2.9: Thermogravimetric Analysis (TGA)
16.2.10: Magnetic Measurements
16.2.11: RNA Binding to PEI-MNPs
16.2.12: Nucleic Acid Protection Against Nucleases by PEI-MNPs
16.2.13: Magnetofection
16.2.14: PEI-MNPs/RNA Internalization
16.3: Results and Discussion
16.3.1: Particle Characterization
16.3.2: RNA Characterization and Binding Experiments
16.3.3: Nucleic Acid Protection from Nuclease Degradation
16.3.4: Knockdown of EGFP on MDA-MB-231: Cells
16.4: Conclusions
Chapter 17: Multimodal Polysilsesquioxane Nanoparticles for
Combinatorial Therapy and Gene Delivery in
Triple-Negative Breast Cancer
17.1: Introduction
17.2: Experimental Section
17.2.1: Synthesis of PSilQ and PSilQ(cur) Nanoparticles
17.2.2: Modification of PSilQ(cur) Nanoparticles with Polyethyleneimine (PEI) Polymer
17.2.3: Functionalization of PEI-PSilQ(cur) Nanoparticles with Polyethylene Glycol (PEG) Polymer
17.2.4: Release of PpIX from PSilQ NPs in a Reducing Environment
17.2.5: Measurement of Intracellular Reactive Oxygen Species
17.2.6: Qualitative Analysis of Reactive
Oxygen Species Generation by
Confocal Laser Scanning Microscopy
17.2.7: In vitro Phototoxicity
17.2.8: Fabrication of Alexa488-Labeled dsDNA-Loaded
or GFP-DS RNA-Loaded PEG-PEI-PSilQ(cur) and
PEI-PSilQ(cur) NPs
17.2.9: Evaluation of Cellular Uptake of Alexa488-
Labeled dsDNA-Loaded PEG-PEI-PSilQ(cur) and
PEI-PSilQ(cur) Nanoparticles
17.2.10: Silencing of Green Fluorescent Protein Using
Green Fluorescent Protein (GFP)-DS RNALoaded
PEG-PEI-PSilQ(cur) and PEI-PSilQ(cur)
NPs in MDA-MB-231/GFP Cells
17.2.11: Influence of the Photochemical Internalization
Effect on the Transfection of GFP-DS RNA in
MDA-MB-231/GFP Cells
17.2.12: Statistical Analysis
17.3: Results and Discussion
17.3.1: Synthesis of Redox-Responsive PpIX Silica Derivative 5
17.3.2: Synthesis of PSilQ, PSilQ(cur), PEI-PSilQ(cur), and PEG-PEI-PSilQ(cur) Nanoparticles
17.3.3: Characterization of PSilQ, PSilQ(cur), PEI-PSilQ-(cur), and PEG-PEI-PSilQ(cur) Nanoparticles
17.3.4: Triggered Release via Reductive Environment
17.3.5: Photostability and 1O2 Generation Studies in Solution
17.3.6: Phototherapy and Chemotherapy Using the PSilQ
and PSilQ(cur) Platform to Treat MDA-MB-231
Cells
17.3.7: Measurement of Intracellular Reactive Oxygen
Species Generated by PSilQ and PSilQ(cur)
Nanoparticles
17.3.8: PSilQ(cur) Platform as Efficient Carrier for the Intracellular Transport of Nucleic Acids
17.4: Conclusions
Chapter 18: A Cationic Amphiphilic Co-polymer as a Carrier of Nucleic Acid Nanoparticles (NANPs) for Controlled Gene Silencing, Immunostimulation, and Biodistribution
18.1: Introduction
18.2: Methods
18.2.1: Synthesis of PgP and NANPs
18.2.2: Nuclease Protection Assay of PgP/DNA Duplex Polyplexes
18.2.3: Physical Characterization of NANPs
18.2.4: Fluorescent Microscopy and Cellular Uptake
18.2.5: Specific Gene Silencing and Cell Viability
18.2.6: Immunostimulation in vitro
18.2.7: Hemolysis Assay in vitro
18.2.8: Biodistribution of PgP/NANP Polyplexes after Systemic Injection
18.2.9: Statistics
18.3: Results
18.3.1: PgP/DNA Stability, Binding, and Nuclease Protection Assay
18.3.2: Intracellular Uptake of PgP/RNA or PgP/DNA Complexes
18.3.3: Characterization of PgP/NANP Polyplexes
18.3.4: Gene Silencing with PgP/NANP(GFP or RhoA) Polyplexes
18.3.5: Hemocompatibility and Immunotoxicity of PgP/NANP Polyplexes
18.3.6: Biodistribution of PgP/NANP Polyplexes after Systemic in vivo Administration
18.4: Discussion
18.5: Conclusion
Chapter 19: Cellular Delivery of RNA Nanoparticles
19.1: Introduction
19.2: Functional RNAs
19.2.1: Interfering RNAs
19.2.2: Aptamers
19.2.3: Splicing Modulators
19.2.4: RNA Switches
19.2.5: Ribozymes
19.2.6: Exogenous mRNAs
19.2.7: CRISPR RNAs
19.2.8: RNA/DNA Hybrids
19.3: Rational RNA Nanostructure Design
19.3.1: Manual Design of RNA Nanostructures Based on Naturally Occurring RNA 3D Motifs
19.3.2: Computational Design of RNA Nanostructures Using Naturally Occurring RNA 3D Motifs
19.3.2.1: 3D motif-based nanostructures
19.3.2.2: De novo design of RNA scaffolds
19.3.3: Functionalization of RNA Nanostructures
19.3.4: Hybrid Technology in RNA NPs
19.4: Challenges in Delivering RNAs
19.4.1: Degradation
19.4.2: Stability in Blood
19.4.3: Hepato-Renal Clearance
19.4.4: Toxicity and Immunogenicity
19.4.5: Cellular Uptake
19.4.6: Endosomal Escape
19.5: Delivery Methods for RNA Therapeutics and RNA NPs
19.5.1: “Naked” Delivery
19.5.2: Mediated Delivery
19.5.2.1: Polymers
19.5.2.2: Hydrogels
19.5.2.3: Peptides
19.5.2.4: Dendrimers
19.5.3: Conjugated Delivery
19.5.3.1: Aptamers
19.5.3.2: Inorganic nanoparticles
19.5.3.3: Microsponges
19.5.4: Encapsulated Delivery
19.5.4.1: Cationic lipids
19.5.4.2: Oxime ether lipids
19.5.4.3: Bolaamphiphile surfactants
19.5.4.4: Exosomes
19.5.4.5: Viral delivery
19.6: Prospects in RNA Nanobiology
Chapter 20: Exosome Mediated Delivery of Functional Nucleic Acid Nanoparticles (Nanps)
20.1: Introduction
20.2: Methods
20.2.1: Cell Culture
20.2.2: Assemblies of RNAA/DNA Fibers, RNA Cubes and RNA Rings Targeting GFP and Their Analysis by N
20.2.3: Atomic Force Microscopy (AFM) Imaging of NANP
20.2.4: Nuclease Protection Assay of DNA Duplex
20.2.5: Isolation of Exosomes
20.2.6: Immunoblotting
20.2.7: Nanoparticle Tracking Analysis and Exosomes Labeling for f-NTA
20.2.8: TEM Analysis
20.2.9: Loading of Exosomes with NANPs
20.2.10: RNA Purification and Quantitative Real Time PCR (RT-qPCR) Analysis
20.2.11: Flow Cytometry
20.2.12: Cell Uptake Imaging
20.2.13: Immunostimulation in vitro
20.3: Results
20.3.1: Characterization of Isolated Exosomes
20.3.2: Characterization of NANP Loaded Exosomes and Nuclease Protection Assay
20.3.3: Cellular Uptake of Exosomes Loaded with NANPs
20.3.4: GFP Gene Silencing with Functionalized NANP Loaded Exosomes
20.3.5: Inhibition of NF-kB Pathway
20.3.6: Immunostimulation by Exosome Loaded NANPs
20.4: Discussion
Chapter 21: Combination of Nucleic Acid and Mesoporous Silica Nanoparticles: Optimization and Therapeutic Performance in vitro
21.1: Introduction
21.2: Experimental Section
21.2.1: Synthesis of NANPs
21.2.2: Complexation of NA-MS-NPs
21.2.3: Characterization of NANPs, MSNPs, and NA-MS-NPs
21.2.4: Nuclease Degradation Protection Studies
21.2.5: Competitive Assay to Study the Release of NANPs from NA-MS-NPs
21.2.6: Immune Response by THP1-Dual Cells and HEK-Blue hTLR3 or 7 Cells
21.2.7: Cellular Uptake of NA-MS-NPs
21.2.8: Cellular Uptake and Intracellular Localization of Alexa546-Labeled dsDNA Complexed with MSNP
21.2.9: Specific Gene Silencing
21.2.10: Cytotoxicity of NA-MS-NPs
21.2.11: Evaluation of Combined Therapy: Cell
Viability, Apoptosis, and Quantitative Reverse
Transcription-Polymerase Chain Reaction
(RT-PCR)
21.2.12: Statistical Analysis
21.3: Results
21.3.1: Synthesis and Characterization of MSNPs
21.3.2: Optimization of Nucleic Acid Binding to MSNPs, pH-Dependent Release, and Enzymatic Stability
21.3.3: Formation of NA-MS-NPs and NANPs’ Integrity Studies upon Their Release from NA-MS-NPs
21.3.4: Immunostimulation by NA-MS-NPs in vitro
21.3.5: Cellular Uptake and Colocalization Studies for dsDNA-Loaded MSNPs
21.3.6: Specific Gene Silencing by NA-MS-NPs
21.3.7: Combination Therapy Using fNA-MS-NPs
21.4: Conclusions
Chapter 22: Opportunities, Barriers, and a Strategy for Overcoming Translational Challenges to Therapeutic Nucle
22.1: Introduction
22.1.1: Identified Opportunities to be Explored
22.1.2: Barriers and Strategies for Overcoming Them
22.1.2.1: Valley of death
22.1.2.2: Lack of systematic studies required for regulatory submissions
22.1.2.3: Inefficient communication between stakeholders
22.2: Conclusions and Prospects
22.2.1: Paths Forward
Part III: Dynamic Nucleic Acid Nanoparticles
Chapter 23: Activation of Different Split Functionalities on Re-association of Rna-Dna Hybrids
23.1: Introduction
23.2: Design and in vitro Studies of RNA-DNA Hybrids
23.3: Intracellular Studies of Hybrid Re-association
23.4: Hybrid Delivery and Re-association in vivo
23.5: siRNAs (against HIV and Cancer) and Aptamers
23.6: Methods
23.6.1: RNA and DNA Sequence Design
23.6.2: Hybrid RNA-DNA Duplex Assemblies and Native PAGE
23.6.3: Recombinant Human Dicer Assay
23.6.4: Human Serum Degradation Studies
23.6.5: Fluorescence Studies
23.6.6: Transfection of Human Cell Lines
23.6.7: Microscopy
23.6.8: Endosomal Co-localization Studies
23.6.9: Flow Cytometry Experiments
23.6.10: In vivo Experiments
23.6.11: HIV-1 Inhibition by RNA-DNA Hybrids
23.6.12: Transfection Experiments with Anti-GSTP1 RNA-DNA Hybrids and Immunoblotting
Chapter 24: Co-transcriptional Production of Rna–Dna Hybrids for Simultaneous Release of Multiple Split Functionalities
24.1: Introduction
24.2: Materials and Methods
24.2.1: RNA and DNA Sequences
24.2.2: Hybrid RNA-DNA Duplexes Assemblies and Native PAGE
24.2.3: Re-association and Recombinant Human Dicer Assay
24.2.4: Release of Malachite Green Aptamer
24.2.5: FRET Studies
24.2.6: Transfection of Human Breast Cancer Cells
24.2.7: Interferon Activation Assay
24.2.8: Microscopy
24.2.9: Flow Cytometry Experiments
24.2.10: Co-transcriptional Production of RNA-DNA Hybrids
24.3: Results
24.3.1: Rational Design of Hybrids and Nomenclature
24.3.2: Hybrid Re-association and Release of Split Functionalities
24.3.3: Intracellular Re-association of Hybrids
24.3.4: Optimization of Hybrids Design
24.3.5: Co-transcriptional Production of RNA-DNA Hybrids
24.4: Discussion
Chapter 25: Triggering of RNA Interference with RNA-RNA, RNA-DNA, and DNA-RNA Nanoparticles
25.1: Introduction
25.2: Results and Discussion
25.2.1: RNA Nanocubes
25.2.2: Functional RNA Nanocubes against HIV-1
25.2.3: RNA-DNA Nanocubes
25.2.4: DNA-RNA Nanocubes
25.2.5: Activation of Interferons by Functional Nanoparticles
25.3: Conclusion
25.4: Methods
25.4.1: 3D Modeling of Functional RNA and DNA Cube Assemblies
25.4.2: Functional RNA and DNA Cube Assemblies and Native PAGE
25.4.3: Dynamic Light Scattering (DLS) Experiments
25.4.4: Recombinant Human Dicer Assay
25.4.5: Human Blood Serum Degradation Studies
25.4.6: Fluorescence Studies
25.4.7: Transfection of Human Breast Cancer Cells
25.4.8: Endosomal Co-localization Studies
25.4.9: Microscopy
25.4.10: Flow Cytometry Experiments
25.4.11: Virus Production
25.4.12: Infectivity Assay
25.4.13: Western Blot Analysis
25.4.14: Reporter-Cell Line for Analysis of Interferon Activation
25.4.15: Primary Human Peripheral Blood Mononuclear
Cell (PBMC) Culture for Analysis of Interferon
and Cytokine Secretion
Chapter 26: Multistrand Structure Prediction of Nucleic Acid Assemblies and Design of RNA Switches
26.1: Introduction
26.2: Methods
26.2.1: Computational Approaches: Algorithm for Prediction of Multistrand RNA Secondary Structures
26.2.2: Representation of Secondary Structures
26.2.3: Search Algorithm
26.2.4: Kinetic Considerations
26.2.5: Sequence Design
26.2.6: Creation of a Three-Dimensional (3D) Model
26.2.7: RNA Test Set
26.2.8: Experimental Approaches: Preparation and Reassociation of Various RNA/DNA Hybrids
26.2.9: Preparation of RNA Switches
26.2.10: Nondenaturing PAGE Experiments
26.2.11: Recombinant Human Dicer Assay
26.2.12: Transfection Experiments in Cultured Human Breast Cancer Cells
26.2.13: Flow Cytometry
26.2.14: Microscopy
26.3: Results and Discussion
26.3.1: Computational Results for Test Set of Single-Sequence RNAs
26.3.2: Reassociation of RNA/DNA Hybrids
26.3.3: Computational Results for RNA Switches
26.3.4: In vitro Studies of RNA Switches
26.3.5: Cell Culture Studies of CTGF-eGFP Switch
Chapter 27: The Use of Minimal RNA Toeholds to Trigger the Activation of Multiple Functionalities
27.1: Introduction
27.2: Rational Design of RNA-DNA Hybrids
27.3: Reassociation of RNA-DNA Hybrids
27.4: Reassociation of RNA-DNA Hybrids in Human Cells
27.5: Controlled Activation of RNAi by Nanorings Functionalized with RNA-DNA Hybrids
27.6: Computational Studies of RNA-DNA Hybrid Reassociation
27.7: Methods
27.7.1: RNA/DNA Nanoparticles and Hybrid Assemblies and Native PAGE
27.7.2: FRET Studies
27.7.3: Transfection of Human Breast Cancer Cells MDA-MB-231
27.7.4: Confocal Microscopy
27.7.5: Flow Cytometry Experiments
27.7.6: HIV-1 Experiments
27.7.7: In silico Predictions of RNA-DNA Hybrid Reassociation
Chapter 28: Functionally-Interdependent Shape-Switching Nanoparticles with Controllable Properties
28.1: Introduction
28.2: Materials and Methods
28.2.1: Nanoparticle Assembly and Purification
28.2.2: UV-Melting Experiments
28.2.3: Kinetics of Re-association Determination
28.2.4: Primary Human Peripheral Blood Mononuclear
Cell and Whole Blood Culture for Analysis of
Interferon and Cytokine Secretion
28.2.5: Nuclease Digestion Assays
28.2.6: Computational Predictions and 3D Modeling
28.2.7: Fluorescence Studies
28.2.7.1: Activation of FRET
28.2.7.2: Activation of Broc-Coli Aptamers
28.2.8: Atomic Force Microscopy (AFM) Imaging and Sample Preparation
28.2.9: Transfection of Human Cell Lines
28.2.10: Fluorescent Microscopy
28.2.11: Flow Cytometry
28.2.12: Cell Proliferation Assay
28.2.13: Statistics
28.3: Results and Discussion
28.3.1: Complementary Nanoparticles have Controlled
Rates of Re-association and Fine-Tunable
Thermodynamic, Chemical and Immunological
Properties
28.3.2: Re-association of Complementary DNA
Nanoparticles Triggers Co-transcriptional
Formation of RNA Nanoparticles
28.3.3: Re-association of Complementary DNA
Nanoparticles Triggers Activation of Embedded
Split RNA Aptamers
28.3.4: Re-association of Complementary Nanoparticles
Triggers Activation of Energy Transfer and RNA
Interference in Cells
28.3.5: Other Examples of Complementary Nanoparticles
28.4: Conclusion
Chapter 29: Dynamic Behavior of RNA Nanoparticles Analyzed by AFM on a Mica/Air Interface
29.1: Introduction
29.2: Materials and Methods
29.2.1: Molecular Dynamics Simulations
29.2.2: Preparation and Assembly of RNA Nanoparticles
29.2.3: Dynamic Light Scattering (DLS)
29.2.4: Transfection of Human Cell Lines
29.2.5: Fluorescence Microscopy
29.2.6: Immunology
29.2.7: AFM Sample Preparation
29.2.8: AFM Imaging and Image Analysis
29.2.9: Computer-Assisted AFM Image Analysis
29.3: Results and Discussion
29.3.1: Molecular Dynamics Simulations
29.3.2: Conventional Characterization of RNA Nanoparticles
29.3.3: Immunological Studies of RNA Nanoparticles
29.3.4: Atomic Force Microscopy (AFM) and Image Analysis
Chapter 30: Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology
30.1: Introduction
30.2: Results and Discussion
30.3: Materials and Methods
30.3.1: Design of Broccoli Fluorets
30.3.2: RNA Preparation
30.3.3: Broccoli Aptamer and Fluoret Assembly
30.3.4: Co-transcriptional Assembly
30.3.5: Electrophoretic Mobility Shift Assays
30.3.6: EDTA Degradation and Mg2+ Formation
30.3.7: Nuclease-Driven Assembly/Degradation
30.3.8: Strand Displacement
30.3.9: Thermal Deactivation/Activation
30.3.10: Blood Stability
30.3.11: Statistics
30.4: Conclusions
Chapter 31: Aptamers as Modular Components of Therapeutic Nucleic Acid Nanotechnology
31.1: Introduction
31.2: Generation of Aptamers via SELEX (Systematic Evolution of Ligands by Exponential Enrichment)
31.3: Aptamers versus Antibodies
31.4: Organization of Aptamers into Complex Structures
31.5: Mechanisms of Action for Stand-Alone Therapeutic Aptamers
31.6: Chimeric Aptamers to Deliver TNAs
31.7: Aptamers as Modular Components of Nucleic Acid Nanoparticles
31.8: Use of Aptamers in Nanorobot Construction
31.9: Light-Up Aptamers as Trackers of NANP Assembly
31.10: Light-Up Aptamers as Trackers for Intracellular Activation of NANPs
31.11: Aptamer-Assisted Packaging, Visualization, and Delivery of NANPs Using Natural Pathways
Part IV: Immunomodulation with NANPs
Chapter 32: Programmable Nucleic Acid Based Polygons with Controlled Neuroimmunomodulatory Properties for Predictive QSAR Modeling
32.1: Introduction
32.2: Results and Discussion
32.3: Conclusion
32.4: Experimental Section
32.4.1: Assembly of Polygons and Their Characterization
32.4.2: 3D Modeling
32.4.3: AFM
32.4.4: DLS
32.4.5: Degradation Assay in Fetal Bovine Serum (FBS)
32.4.6: Equilibrium Dissociation Constant (KD) Measurements
32.4.7: Structural Integrity of Polygons Associated with Lipofectamine 2000 (L2K)
32.4.8: Transfections
32.4.9: Cell Viability
32.4.10: Relative Uptake Efficiencies in hHm
32.4.11: ELISA
32.4.12: Statistics
32.4.13: LAL Assay
32.4.14: QSAR Modeling: Data Set
32.4.15: QSAR Approach
32.4.16: Descriptors
32.4.17: Machine Learning Method: RF
32.4.18: Model Construction and Validation
32.4.19: Evaluation of the Model Prediction Accuracy
Chapter 33: Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles
33.1: Introduction
33.2: Assembly and Characterization of NANPs
33.3: NANPs Require a Delivery Carrier to Induce Interferon Response
33.4: NANP Structure and Composition Define Immunorecognition
33.5: PBMC Internalize NANPs via the Endolysosomal Pathway
33.6: pDCs Recognize NANPs and TLR7 Contributes to IFN Induction
33.7: Conclusions
33.8: Materials and Methods
33.8.1: NANP Synthesis and Characterization
33.8.2: Primary Human PBMC Isolation
33.8.3: Stimulation of PBMCs with DNA and RNA Nanoparticles for Cytokine Induction
33.8.4: Purification and Culture of DC Subsets for Nanoparticle Stimulation
33.8.5: Characterization of Nanoparticle Association
with PBMCs by Flow Cytometry and Confocal
Microscopy
33.8.6: Inhibitor Studies
33.8.7: Reporter Cell Culture and Experiments
33.8.8: Statistics
Chapter 34: RNA Fibers as Optimized Nanoscaffolds for siRNA Coordination and Reduced Immunological Recognition
34.1: Introduction
34.2: Results and Discussion
34.3: Experimental Section
34.3.1: Molecular Dynamics Simulations
34.3.2: Fibers Preparation and Assembly
34.3.3: Blood Stability Assays
34.3.4: Atomic Force Microscopy
34.3.5: UV-Melting Experiments
34.3.6: In vitro Immunology
34.3.7: Transfection of Human Cell Lines and Gene Silencing Experiments
34.3.8: Fluorescence Microscopy
34.3.9: Preparation of PEG-MSNs
34.3.10: Optimization of PEC-MSNs for Gene Delivery
34.3.11: Transmission Electron Microscopy
34.3.12: Dynamic Light Scattering
34.3.13: Cellular Uptake of PEG-MSN-RNA Fiber
34.3.14: Gene Silencing in vitro with PEG-PEI-MSN as Transfection Agent
34.3.15: Statistics
Chapter 35: RNA–DNA fibers and polygons with Controlled immunorecognition Activate RNAi, FRET and Transcriptional Regulation of NF-kB in Human Cells
35.1: Introduction
35.2: Materials and Methods
35.2.1: Design of RNA-DNA Fibers and Polygons
35.2.2: Assemblies of Hybrid RNA-DNA Fibers and Polygons and Their Analysis by Native-PAGE
35.2.3: Ultraviolet Melting Experiments
35.2.4: Kinetics of Reassociation
35.2.5: Blood Stability
35.2.6: Atomic Force Microscopy (AFM) Imaging
35.2.7: Primary Human Peripheral Blood Mononuclear Cells (PBMCs) and Whole-Blood Culture for Analysis of Interferon and Cytokine Secretion
35.2.8: Reporter Cell-Based Assay
35.2.9: Activation of FRET
35.2.10: Transfection of Human Breast Cancer Cells Expressing Green Fluorescent Protein (MDA-MB-231/GFP
35.2.11: Analysis of Cell Death and Cell Cycle by Propidium Iodide Staining and Flow Cytometry
35.2.12: Western Blot
35.2.13: Immunofluorescence Analysis for Detection of NF-kB
35.2.14: Statistical Analysis
35.3: Results and Discussion
Chapter 36: Toll-Like Receptor-Mediated Recognition of Nucleic Acid Nanoparticles (NANPs) in Human Primary Blood Cells
36.1: Introduction
36.2: Results and Discussion
36.2.1: Plasmacytoid Dendritic Cells as Main Responders to NANPs in Human PBMC
36.2.2: TLR7 and TLR9 Involved in NANPs Recognition by Human PBMC
36.2.3: Electroporation Suppresses TLR9: Functionality in Human PBMC without Affecting Cell Viability
36.3: Materials and Methods
36.3.1: Reagents
36.3.2: NANPs Synthesis and Characterization
36.3.3: Primary Human Peripheral Blood Mononuclear Cell (PBMC) Isolation and Treatment with NANPs
36.3.4: Electroporation of PBMCs with Nucleic Acid Nanoparticles
36.3.5: Western Blot Analysis of TLR Expression
36.3.6: siRNA Delivery
36.3.7: Statistical and Data Analysis
36.4: Summary and Conclusions
Chapter 37: Smart-Responsive Nucleic Acid Nanoparticles (NANPs) with the Potential to Modulate Immune Behavior
37.1: Introduction
37.2: Dynamic Shape-Switching and Functional Activation with NANPs
37.2.1: Activation of RNA Interference, FRET, RNA Aptamers, and Transcription Initiation
37.2.2: Fine-Tunable Properties
37.3: Immunostimulatory Properties of NANPs
37.4: Conclusions
Chapter 38: Innate Immune Responses Triggered by Nucleic Acids Inspire the Design of Immunomodulatory Nucleic Acid Nanoparticles (NANPs)
38.1: Introduction
38.2: Recognition Receptors
38.3: Signature Motifs
38.4: Safety Considerations and Future Directions
Chapter 39: Retinoic Acid Inducible Gene-I Mediated Detection of Bacterial Nucleic Acids in Human Microglial Cells
39.1: Introduction
39.2: Materials and Methods
39.2.1: Source and Propagation of Human Glial Primary Cells and Cell Lines
39.2.2: Murine Glial Cell Isolation and Culture
39.2.3: Bacterial Propagation
39.2.4: Bacterial Infection
39.2.5: Nuclear translocation
39.2.6: Immunoblot Analysis
39.2.7: Quantification of Cytokines in Glial Cell Supernatants
39.2.8: Ligand Stimulation
39.2.9: Transfection
39.2.10: BX795 Treatment
39.2.11: siRNA Knockdown
39.2.12: Nucleic Acid-Based Nanoparticles Assembly
39.2.13: Atomic Force Microscopy Imaging
39.2.14: Statistical Analysis
39.3: Results
39.3.1: Microglia Show Upregulated RIG-I Protein Expression Following Bacterial Infection
39.3.2: Microglia Show Upregulated RIG-I Protein Expression in Response to Bacterial Components
39.3.3: Bacterial Components Stimulate IRF3 Phosphorylation in Human Microglia
39.3.4: Bacterial Nucleic Acids Stimulate RIG-I-Dependent Interferon Responses in Human Microglia
39.3.5: Nucleic Acid Nanoparticles Stimulate RIG-I-Dependent Responses in Human Microglia
39.4: Discussion
39.5: Conclusions
Chapter 40: Use of Human Peripheral Blood Mononuclear Cells to Define Immunological Properties of Nucleic Acid Nanoparticles
40.1: Introduction
40.1.1: Nucleic acid-Based technologies
40.1.2: Therapeutic NANPs
40.1.3: NANPs’ Safety and Immune-Mediated Efficacy Considerations
40.1.4: Development of the Protocol
40.1.5: Advantages
40.1.6: Limitations
40.1.7: Overview of the Procedure
40.1.8: Applications
40.1.9: Alternative Methods
40.1.10: Experimental Design
40.1.10.1: PBMCs
40.1.10.2: Controls
40.1.10.3: Preparation of nucleic acid components
40.1.10.4: NANP design and assembly
40.1.10.5: Cytokine detection
40.1.10.6: Understanding the mechanism of NANP recognition
40.2: Materials
40.2.1: Biological Materials
40.2.2: Reagents
40.2.2.1: ELISA kits
40.2.3: Equipment
40.2.4: Reagent Setup
40.2.4.1: 5X NANP assembly buffer
40.2.4.2: 1X native-PAGE loading buffer
40.2.4.3: 1X native-PAGE running buffer
40.2.4.4: Complete RPMI 1640 medium
40.2.4.5: Lipopolysaccharide for positive control in proinflammatory cytokine and chemokine analysis
40.2.4.6: Phytohemagglutinin for positive control in type II interferon analysis
40.2.4.7: ODN2216 for positive control in type I and type III interferon analysis
40.2.4.8: ODN2088 for mechanistic study to reveal potential involvement of endosomal TLRs
40.2.4.9: Fucoidan, dextran sulfate, poly-I, chondroitinsulfate, and poly-C preparation for mechanisticstudies
40.2.4.10: Heat-inactivated fetal bovine serum
40.3: Procedure
40.3.1: Assembly of NANPs
40.3.2: Verification of NANP Assemblies by Native-PAGE
40.3.3: Purification of NANPs by Native-PAGE
40.3.4: Assessment of Structural Integrity of the NANPs upon Complexation with Delivery Reagents
40.3.5: Isolation of PBMCs
40.3.6: Exposing PBMCs to NANPs and Collecting Supernatants
40.3.7: Detection of Biomarkers
40.3.8: Mechanistic Insight into NANP Uptake Routes and Immune Receptor Recognition
40.4: Troubleshooting
40.5: Timing
40.6: Anticipated Results
40.6.1: Anticipated Results for NANP Assembly and Retention of NANP Structural Integrity upon Interaction with Transfection Reagents
40.6.2: Anticipated Results for Endotoxin Screening (Step 19)
40.6.3: Anticipated Results for Cytokine Screening (Steps 32-57)
40.6.4: Anticipated Results for Mechanistic Analysis
Chapter 41: The Immunorecognition, Subcellular Compartmentalization, and Physicochemical Properties of Nucleic Acid Nanoparticles can be Controlled by Composition Modification
41.1: Introduction
41.2: Materials and Methods
41.2.1: NANP Synthesis
41.2.2: UV-Melting Experiments
41.2.3: Dynamic Light Scattering Analysis
41.2.4: Fetal Bovine Serum Stability Assay
41.2.5: Integrity of NANP upon Release from a Carrier
41.2.6: Source and Propagation of Cell Lines
41.2.7: Reporter Cell Lines
41.2.8: Transfection of Microglia
41.2.9: siRNA Knockdown
41.2.10: Quantification of Cytokines in Cell Supernatants
41.2.11: Immunoblot Analyses
41.2.12: Cellular Fractionation
41.2.13: Flow Cytometric Analysis
41.2.14: Fluorescent Immunohistochemical Analysis
41.2.15: Statistical Analysis
41.3: Results and Discussion
41.3.1: NANP Composition Defines Their Physicochemical Properties
41.3.2: Carrier and NANP Composition Affect Internalization and Subcellular Localization
41.3.3: Composition Affects NANP-Induced Cytokine Production Mediated by NF-kB and IRF
41.3.4: NANP Composition Affects PRR Activation
41.4: Conclusion
Index