Riboregulator Design and Analysis

Preface
Contents
Contributors
Chapter 1: RNP-Based Control Systems for Genetic Circuits in Synthetic Biology Beyond CRISPR
1 Introduction
2 Current Systems
2.1 Engineered RNPs in Prokaryotes
2.1.1 RNA-Binding Protein (RBP)-RNA Regulated Systems
2.1.2 CRISPR-Cas-Regulated Systems
2.2 Engineered RNPs in Eukaryotes
2.2.1 Engineered RNA-Binding Protein-RNA-Regulated Systems
2.2.2 CRISPR-Cas-Regulated Systems
3 Selecting an Appropriate System
4 Future Perspectives
References
Chapter 2: Computational Design of RNA Toehold-Mediated Translation Activators
1 Introduction
1.1 Toehold Switches
1.2 SNIPRs
2 Materials
3 Methods
3.1 Installation of Software Packages
3.2 Computational Design of Toehold Switches
3.3 Computational Design of SNIPRs
4 Notes
References
Chapter 3: Design of RNA-Based Translational Repressors
1 Introduction
2 Materials
2.1 Toehold Repressor and Three-Way Junction (3WJ) Repressor
2.2 Suitable Vectors
2.3 Cloning
2.4 Chemically Competent E. coli
2.5 Strains and Growth Condition
2.6 Plate Reader
2.7 Flow Cytometry
3 Methods
3.1 Design of RNA Translational Repressors
3.2 Cloning of RNA Translational Repressor Expression Vectors
3.3 Making of Chemically Competent E. coli for Cloning and Experimental Transformant
3.4 Experiments with RNA Translational Repressors
4 Notes
References
Chapter 4: Design of Ribocomputing Devices for Complex Cellular Logic
1 Introduction
2 Materials
2.1 Toehold Switch Elements and Sample Two-Input Ribocomputing Devices
2.2 Suitable Vectors
3 Methods
3.1 Introduction to NUPACK Nucleic Acid Sequence Design
3.2 Design of Orthogonal Toehold Switch Libraries
3.3 Design of Multi-input AND Logic Devices
3.4 Design of Multi-input OR Logic Devices
3.5 Design of A AND (NOT B) Logic Devices
4 Notes
References
Chapter 5: Computational Design of Small Transcription Activating RNAs (STARs)
1 Introduction
2 Materials
2.1 Computational STAR Design
2.2 Construction of STAR Plasmids Using Inverse PCR
2.3 GFP Activation Assays
3 Methods
3.1 Computational STAR Design
3.2 Construction of the STAR Plasmids Using Inverse PCR
3.3 GFP Activation Assays
4 Notes
References
Chapter 6: Design and Assembly of Multilevel Transcriptional and Translational Regulators for Stringent Control of Gene Expres...
1 Introduction
2 Materials
2.1 Creation of New Toolkit Compatible Parts
2.2 Reagents for MLC Assembly
3 Methods
3.1 Part Creation by DNA Synthesis
3.2 Part Creation Through PCR Amplification
3.3 Part Creation Using Annealed DNA Oligos
3.4 MLC Assembly
4 Notes
References
Chapter 7: Model-Based Design of Synthetic Antisense RNA for Predictable Gene Repression
1 Introduction
2 Materials
2.1 asRNA Design
2.2 asRNA Plasmid Construction
2.3 GFP Repression Assays
3 Methods
3.1 Model-Based asRNA Design
3.2 Construction of the asRNA Plasmid Using Inverse PCR
3.3 GFP Repression Assays
4 Notes
References
Chapter 8: Design of a Toolbox of RNA Thermometers
1 Introduction
2 Materials
3 Methods
3.1 Design Concept
3.2 Computational Assessment
3.3 Experimental Synthesis
3.4 Experimental Measurement in a Cell-Free Expression System
3.5 Experimental Measurement in Cells
4 Notes
References
Chapter 9: Development of Synthetic Riboswitches to Guide the Evolution of Metabolite Production in Microorganisms
1 Introduction
2 Materials
2.1 Preparation of RNA Aptamer Library
2.2 Preparation of Ligand-Coupled Agarose Bead for RNA Aptamer Selection
2.3 RNA Aptamer SELEX
2.4 Riboswitch Library Preparation
2.5 In Vivo Selection of Riboswitch Library
2.6 In Vivo Riboswitch Library Validation
2.7 Oligonucleotides
3 Methods
3.1 Preparation of RNA Aptamer Library
3.2 Preparation of Ligand-Coupled Agarose Bead for RNA Aptamer Selection
3.3 RNA Aptamer SELEX
3.4 Riboswitch Library Preparation
3.5 In Vivo Selection of Riboswitch Library
3.6 In Vivo Riboswitch Library Validation and Characterization
4 Notes
References
Chapter 10: Efficient Method to Identify Synthetic Riboswitches Using RNA-Based Capture-SELEX Combined with In Vivo Screening
1 Introduction
2 Materials
3 Capture-SELEX
3.1 Buffers and Solutions
3.2 Initial RNA Pool Preparation
3.3 Hybridization of RNA Pool and Capture Oligonucleotide
3.4 Magnetic Bead Preparation
3.5 Reverse Transcription (RT) PCR
3.6 In Vitro Transcription and Radioactive Labelling
4 In Vivo Screening in Saccharomyces cerevisiae
4.1 Buffers, Solutions, and Media
4.2 Consumables
4.3 Plasmids and Primers
4.4 Enzymes
4.5 Strains
5 Methods
5.1 Capture-SELEX
5.2 Initial RNA Pool Preparation
5.3 Hybridization of RNA Pool and Capture Oligonucleotide
5.4 Preparation of Magnetic Beads and Pool Immobilization
5.5 Selection Process: Removal of Weak Binders and Elution of Ligand Binding Aptamers
5.6 Quantification (Radioactive Measurement Using a Scintillation Counter)
5.7 Amplification of the Enriched Pool
5.8 In Vitro Transcription and Radioactive Body Labelling
6 In Vivo Screening in S. cerevisiae
6.1 Preparation of the Pool for Use as Insert
6.2 Preparation of Plasmid
6.3 Preparation of Electrocompetent Yeast Cells and Transformation
6.4 Screening Process
6.5 Passage Through Escherichia coli and Sequencing of Candidates
6.6 Retransformation of S. cerevisiae with Pure Clones of Candidates and Remeasurement
6.7 Further Steps
7 Notes
References
Chapter 11: RNA Design Principles for Riboswitches that Regulate RNase P-Mediated tRNA Processing
1 Introduction
1.1 Design Strategy
1.1.1 Model Development
1.1.2 Model Implementation, Sequence Sampling, and Scoring
1.2 Biochemical Evaluation of Riboswitch Functionality and the Switching Mechanism
1.2.1 In Vivo Analysis
1.2.2 Kinetic Analysis According to Michaelis-Menten
1.2.3 Structure Analysis by In-Line Probing
1.2.4 Interpretation of the Biochemical Data
2 Materials
2.1 Applied Software
2.2 Fluorescence Measurements
2.3 In Vitro Transcription and RNA Purification of Substrate and M1 RNA
2.4 Purification of RNA by Gel Electrophoresis
2.5 5′-Dephosphorylation of RNA
2.6 5′-Labeling of DNA Probes for Northern Blot or RNA for In-Line Probing
2.7 RNA Extraction and Northern Blot
2.8 Kinetic Analysis
2.9 Structural Analysis by In-Line Probing
3 Methods
3.1 In Silico Design of a New Riboswitch Mechanism
3.2 Fluorescence Measurements
3.3 In Vitro Transcription
3.4 Purification of RNA by Gel Electrophoresis
3.5 5′-Dephosphorylation and 5′-Labeling of RNA
3.6 5′-Labeling of DNA Probes
3.7 RNA Extraction for Northern Blot Analysis
3.8 Northern Blot
3.9 Kinetic Analysis
3.10 Structural Analysis by In-Line Probing
4 Notes
References
Chapter 12: Design, Characterization, and Application of Targeted Gene Activation in Bacteria Using a Modular CRISPRa System
1 Introduction
2 Materials
2.1 Identification of Target Sites and sgRNA Design
2.2 sgRNA Cloning
2.3 Gene Activation Experiments
2.4 Expanding Gene Activation Range
2.5 Evaluating Activation Patterns for CRISPRa Systems
3 Methods
3.1 Identification of Target Sites and sgRNA Design
3.2 sgRNA Cloning
3.3 Gene Activation Experiments
3.4 Expanding Gene Activation Range
3.5 Evaluating Activation Patterns for CRISPRa Systems
4 Notes
References
Chapter 13: Reprogramming TracrRNAs for Multiplexed RNA Detection
1 Introduction
2 Materials
2.1 Reagents and Consumables
2.2 Equipment
3 Methods
3.1 Design of Sensed RNA, Rptr, and Targeted Plasmid for In Vitro RNA Detection
3.2 In Vitro RNA Transcription
3.3 In Vitro DNA Cleavage Assay
3.4 Design and Construction of Rptr and Targeted Plasmids for RNA Detection In Vivo
3.5 E. coli Transformation
3.6 Induction and In vivo Detection
4 Notes
References
Chapter 14: Harnessing CRISPR-Cas9 for Epigenetic Engineering
1 Introduction
1.1 CRISPR-Cas9-Based Epigenome Editing Tools
1.2 Strategies for Optimized Epigenome Editing in Mammalian Cells
2 Materials
2.1 HEK293T Cell Culture
2.2 Guide Cloning
2.3 Transient Transfection
2.4 Chromatin Immunoprecipitation (ChIP)
3 Methods
3.1 Guide Cloning in Destination Vector
3.2 Transient Transfection of Expression Vector for CRISPR/dCas9-Based Epigenetic Effector and gRNA Plasmid with Lipofectamine...
3.3 Chromatin Immunoprecipitation (ChIP)
4 Notes
References
Chapter 15: RNA Structure Prediction, Analysis, and Design: An Introduction to Web-Based Tools
1 Introduction
2 RNA Secondary Structure Prediction
2.1 Structure Prediction of Single Sequences via Free Energy Minimization
2.2 Prediction of Pseudoknots
2.3 Incorporating Experimental Data
2.4 Conserved Secondary Structure Prediction
2.5 Comparing Folding Tools
3 RNA-RNA Interactions
3.1 Predicting RNA-RNA Interactions
3.2 Applications of RNA-RNA Interaction Prediction
4 Designing RNA Structures
5 Conclusion
References
Chapter 16: Single-Molecule FRET Studies of RNA Structural Rearrangements and RNA-RNA Interactions
1 Introduction
2 Materials
2.1 RNA Substrate Design and Preparation
2.2 mRNA-sRNA Annealing
2.3 Ligation of Long mRNA with Internal Fluorophores
2.4 mRNA Structural Rearrangements During sRNA-mRNA Annealing
2.5 Total Internal Reflection Microscopy
2.6 Data Analysis
3 Methods
3.1 Design and Preparation of RNAs for Single-Molecule Experiments
3.2 mRNA-sRNA Annealing
3.3 Visualizing mRNA Structural Rearrangements During sRNA-mRNA Annealing
3.4 Data Analysis: Initial Data Processing
3.5 Analysis of sRNA-mRNA Annealing Kinetics: Binding Lifetimes and FRET Efficiency
3.6 Analysis of mRNA Restructuring: FRET States
4 Notes
References
Chapter 17: Cotranscriptional RNA Chemical Probing
1 Introduction
1.1 Cotranscriptional RNA Structure Probing
1.2 Limitations of Cotranscriptional RNA Chemical Probing Experiments
1.3 Lessons from the Application of Cotranscriptional RNA Chemical Probing to Riboswitches
1.4 Scope of This Protocol
2 Materials
2.1 Equipment
2.2 DNA Template Preparation
2.3 Pre-adenylation of the 3′ Adapter Oligonucleotide
2.4 In Vitro Transcription and Cotranscriptional RNA Structure Probing
2.5 RNA Purification
2.6 3′ Adapter Ligation
2.7 Denaturing Polyacrylamide Gel Electrophoresis
3 Methods
3.1 In Vitro Transcription DNA Template Design
3.2 Choosing an RNA Polymerase Roadblocking Strategy
3.3 Preparation of Randomly Biotinylated Template DNA
3.4 Pre-adenylation of the 3′ Adapter Oligonucleotide
3.5 Single-Round In Vitro Transcription and Cotranscriptional RNA Structure Probing
3.6 RNA Purification
3.7 3′ Adapter Ligation
3.8 Options for Illumina Sequencing Library Construction
3.9 Denaturing PAGE to Assess Single-Round In Vitro Transcription Reaction Products
4 Notes
References
Index