RNA Scaffolds: Methods and Protocols

Preface
	Contents
Contents
Contributors
Chapter 1: Predicting RNA Scaffolds with a Hybrid Method of Vfold3D and VfoldLA
	1 Introduction
	2 Algorithms
		2.1 RNA Secondary Structural Motifs and Single-Stranded Loops
		2.2 RNA Motif-based Template Library
		2.3 RNA Loop-Based Template Library
		2.4 Sequence Similarity-Based Score
	3 Methods
	4 Notes
	References
Chapter 2: RNA Footprinting Using Small Chemical Reagents
	1 Introduction
		1.1 Footprinting with DMS or SHAPE Reagent
		1.2 RNA Retrieval and Sequencing
	2 Materials
		2.1 Footprinting with DMS and SHAPE Reagent
		2.2 RNA Retrieval and Sequencing
	3 Methods
		3.1 Footprinting with DMS and SHAPE Reagents
		3.2 RNA Retrieval and Sequencing
		3.3 Data Analysis
		3.4 Troubleshooting
	4 Notes
	References
Chapter 3: Improving RNA Crystal Diffraction Quality by Postcrystallization Treatment
	1 Introduction
	2 Materials
	3 Methods
		3.1 Design and Synthesis of T-Box RNA and tRNA for Crystallization
		3.2 Crystallization of the T-Box Stem I-tRNA-YbxF Ternary Complex
		3.3 Postcrystallization Treatments
		3.4 Understanding the Basis of Treatment-Induced Improvement of Crystal Quality
	4 Notes
	References
Chapter 4: Using tRNA Scaffold to Assist RNA Crystallization
	1 Introduction
	2 Materials
	3 Methods
		3.1 Design and Synthesize tRNA Scaffold Vector
		3.2 tRNA-RNA Chimera Plasmid Construction
		3.3 tRNA-RNA Sample Preparation
	4 Notes
	References
Chapter 5: RNA Modeling with the Computational Energy Landscape Framework
	1 Introduction
	2 Algorithms
		2.1 Selecting Pairs of Minima for Transition state Searches
		2.2 Transition State Searches
		2.3 Search Strategies
		2.4 Visualization of the Energy Landscape
		2.5 Available Potential Energy Models
		2.6 Analysis of Structural Ensembles
	3 Method
		3.1 Starting Points
		3.2 Exploration of the Energy Landscape
		3.3 Thermodynamics
		3.4 Example Application
	4 Notes
	References
Chapter 6: Coexpression and Copurification of RNA-Protein Complexes in Escherichia coli
	1 Introduction
	2 Materials
		2.1 RNA-Protein Expression
		2.2 RNA/Protein Purification
	3 Methods
		3.1 Cell Growth
		3.2 RNA-Protein Complex Purification
	4 Notes
	References
Chapter 7: In Vivo Production of Small Recombinant RNAs Embedded in 5S rRNA-Derived Protective Scaffold
	1 Introduction
	2 Materials
		2.1 Bacterial Strain and Plasmid Vector
		2.2 Equipment
		2.3 Supplies
		2.4 Reagents
			2.4.1 General Purpose Reagents
			2.4.2 Cloning of the Recombinant RNA Coding Sequence into pCA2c Plasmid Vector
			2.4.3 Plasmid Purification
			2.4.4 Bacterial Cells Growth
			2.4.5 Cell Lysis and Fractionation of Nucleic Acids
			2.4.6 Preparative Electrophoresis in Denaturing Polyacrylamide  Gel
			2.4.7 Agarose Gel Electrophoresis
			2.4.8 RNA Cleavage with DNAzymes
		2.5 Software
	3 Methods
		3.1 Guidelines for Designing the Chimeric RNA and DNAzymes
		3.2 Construction of the RNA Expression Vector and Transformation of the E. coli Cells
		3.3 Cultivation and Harvesting of the Transformed E. coli Cells
		3.4 Cell Lysis and Crude Fractionation of Nucleic Acids
		3.5 Purification of the Chimeric RNA by Preparative Electrophoresis
		3.6 DNAzyme Cleavage of the Chimeric RNA
		3.7 Separation of the Excised Cargo RNA from Other Components of the DNAzyme Cleavage Reaction Mixture
	4 Notes
	References
Chapter 8: Production of Circular Recombinant RNA in Escherichia coli Using Viroid Scaffolds
	1 Introduction
	2 Materials
		2.1 Bacterial Strains and Plasmids
		2.2 Culture Media
		2.3 Purification and Analysis of Recombinant RNA
	3 Methods
		3.1 Plasmid Design
		3.2 Plasmid Construction
		3.3 Recombinant RNA Production
		3.4 Purification and Analysis of Recombinant RNA
	4 Notes
	References
Chapter 9: Identification of RNA-Binding Proteins Associated to RNA Structural Elements
	1 Introduction
	2 Materials
		2.1 Pull-down and Cell Extract Preparation Materials
		2.2 RNA Chimera  Prep
		2.3 Cell Extract  Prep
		2.4 RNA-Protein Pull Down
	3 Methods
		3.1 RNA Chimera Purification
		3.2 Preparation of S10 Cell Lysates
		3.3 Pull-down Assay Using Streptavidin-Aptamer Tagged RNA
			3.3.1 Binding of the RNA Chimera to Streptavidin-Coated Magnetic Beads
			3.3.2 Block RNA-Bound Streptavidin Coated Magnetic Beads
			3.3.3 Binding of Proteins to RNA Chimera
		3.4 Mass Spectrometry Identification of RNA-Eluted Factors
	4 Notes
	References
Chapter 10: Live Cell Imaging Using Riboswitch-Spinach tRNA Fusions as Metabolite-Sensing Fluorescent Biosensors
	1 Introduction
	2 Materials
		2.1 Equipment and Supplies
		2.2 Reagents
	3 Method
		3.1 Construction and Transformation of Biosensor and Enzyme Constructs
			3.1.1 Cloning of Biosensor Expression Vector
				Procedure Starting from Original pET31b Vector
					Step 1a-Generation of Vc2-Spinach tRNA Construct (Estimated Time: 2 h)
					Step 2a-Generation of Vc2-Spinach tRNA Construct with T7 Promoter and Restriction Sites (Estimated Time: 2 h).
					Step 3a-Cloning of Biosensor Insert into Expression Vector (Estimated Time: 2 Days)
				Alternative Procedure Starting from Existing Biosensor Construct
					Step 1b-Generation of Biosensor-Spinach Insert
					Step 2b-Cloning of Biosensor Insert into Expression Vector (Estimated time: 1 Day)
			3.1.2 Construction of Enzyme Expression Vector
				Step 4-Generation of WspR Diguanylate Cyclase Insert (Estimated Time: 3 h)
				Step 5-Cloning of WspR Diguanylate Cyclase into Expression Vector (Estimated Time: 2 Days)
			3.1.3 Transformation of Expression Vectors for Live Cell Imaging
				Step 6a-Generation of Strains Containing Both Biosensor and Enzyme Constructs (Estimated Time: 1 Day)
				Step 6b-Alternative Procedure for Generation of Strains Containing Biosensor Only (Estimated Time: 1 Day)
		3.2 Live Cell Imaging of the RNA-Based Biosensor
			3.2.1 Preparation of Poly-d-Lysine Coverslips for Fluorescence Microscopy
				Step 7-Acid Rinse of Coverslips (Estimated Time: 11 h)
				Step 8-Poly-d-Lysine Rinse of Coverslips (Estimated Time: 11 h)
			3.2.2 Fluorescent Microscopy Experiments
				Step 9-Bacterial Growth and Induction Conditions (Estimated Time: 16-20 h)
				Step 10-Harvesting Cells and Preparation of Slides (Estimated Time: 3.5 h)
				Step 11-Imaging Cells Using Fluorescence Microscopy (Estimated Time: 1 h)
				Step 12-Analysis of Fluorescence Microscopy Data (Estimated Time: 3 h)
			3.2.3 Flow Cytometry Experiments
				Step 13a-Growth of Cells with IPTG Induction for Flow Cytometry (Estimated: 16-18 h)
				Step 14a-Preparation of Cells for Flow Cytometry (Estimated Time: 1 h)
				Step 13b-Alternative Procedure for Growth of Cells with Autoinduction Media for Flow Cytometry (Estimated: 42 h)
				Step 14b-Alternate Procedure for Treatment of Cells with Exogenous Analytes for Flow Cytometry (Estimated Time: 25 min)
				Step 15-Analysis of Cells by Flow Cytometry (Estimated Time: 1 h)
				Step 16-Analysis of Flow Cytometry Data (Estimated Time: 1 h)
	4 Notes
	References
Chapter 11: Rational Design of Allosteric Fluorogenic RNA Sensors for Cellular Imaging
	1 Introduction
	2 Materials
		2.1 In Vitro RNA Preparation and Characterization
		2.2 Molecular Cloning
		2.3 Intracellular Imaging
	3 Methods
		3.1 In Silico Design of Fluorogenic RNA-Based Tetracycline Sensors
		3.2 In Vitro Optimization of Fluorogenic RNA-Based Tetracycline Sensors
		3.3 Molecular Cloning of Sensors into E. coli Cells
		3.4 Fluorescence Imaging of Tetracycline in Live E. coli Cells
	4 Notes
	References
Chapter 12: Riboswitch-Mediated Detection of Metabolite Fluctuations During Live Cell Imaging of Bacteria
	1 Introduction
	2 Materials
		2.1 Equipment and Supplies
		2.2 Reagents
	3 Method
		3.1 Construction of a Riboswitch-yfp Reporter
			3.1.1 Assembly of the lchAA Leader-yfp Sequence
			3.1.2 Transformation of B. subtilis
		3.2 Live Cell Imaging of B. subtilis Harboring the lchAA Leader-yfp Reporter
			3.2.1 Growth of Bacterial Strains for Fluorescence Microscopy
			3.2.2 Use of Agarose Pads for Fluorescence Microscopy
			3.2.3 Fluorescence Microscopy Experiments
	4 Notes
	References
Chapter 13: FRET Analysis of RNA-Protein Interactions Using Spinach Aptamers
	1 Introduction
	2 Materials
		2.1 Equipment and Supplies
			2.1.1 RNA and Protein Cloning
			2.1.2 In Vitro Transcription and Purification of RNA
			2.1.3 Expression and Purification of Protein
			2.1.4 REMSAs
			2.1.5 Donor Quenching Assay and Competition Assay
		2.2 Reagents
			2.2.1 RNA and Protein Cloning
			2.2.2 In Vitro Transcription and Purification of RNA
			2.2.3 Expression and Purification of Protein
			2.2.4 REMSAs
	3 Method
		3.1 Construct Design, Subcloning, Synthesis, and Purification Spinach-pp7-RNA Fusion
			3.1.1 Construction of Spinach-pp7-RNA
			3.1.2 Subcloning of Spinach-pp7-RNA
			3.1.3 Synthesis of Spinach-pp7-RNA-In Vitro Transcription and Purification
				Step 1: Linearization of pUC19-Spinach-pp7 (see Subheading 3.1.2, Fig. 2b)
				Step 2a: In Vitro Transcription of Spinach-pp7 RNA
				Step 2b: Reaction Process Monitoring
				Step 3: Purification of In Vitro Transcribed RNA
				Step 4: Quality Check of Purified RNA.
		3.2 Construction and Transformation of mCherry Fused PP7 Constructs
			3.2.1 Construction of mCherry Fused PP7 Proteins
			3.2.2 Cloning of mCherry Tagged PP7 Constructs
			3.2.3 Expression and Purification of PP7 Constructs
				Step 1: Expression of PP7 Fusion Proteins
				Step 2: Cell Lysis
				Step 3a: Purification of His6-Tagged mCherry-PP7
				Alternative Step 2b: Purification of GST-tagged PP7-mCherry, PP7, and mCherry
				Step 3: Size exclusion Chromatography
		3.3 Investigate Direct Binding of RNA-Protein Interactions
			3.3.1 Preparation of Fluorescent Native RNA Electromobility Shift Assay (Fluorescence REMSA)
			3.3.2 Fluorescent Native RNA Electromobility Shift Assay
				Step 1: Sample Preparation
				Step 2: Gel and Sample Preparation and Electrophoresis
				Step 3: DFHBI Staining and Fluorescence Read Out
			3.3.3 Preparation of Homogeneous RNA-Protein FRET-Based Assay
			3.3.4 Homogeneous RNA-Protein FRET-Based Assay
				Step 1: Sample Preparation
				Step 2: Readout
			3.3.5 Homogeneous RNA-Protein FRET-Based Competition Assay
				Step 1: Preformation of the RNA-Protein Complex
				Step 2: Titration of Competitor
				Step 3: Readout
	4 Notes
	References
Chapter 14: Engineering Aptazyme Switches for Conditional Gene Expression in Mammalian Cells Utilizing an In Vivo Screening Ap...
	1 Introduction
	2 Materials
		2.1 Construction of Randomized Aptazyme Libraries
		2.2 Preparation of Randomized Aptazyme Plasmid Libraries
		2.3 Mammalian Cell Culture and Screening
	3 Methods
		3.1 Construction of Randomized Aptazyme Libraries
		3.2 Preparation of Randomized Aptazyme Plasmid Libraries
		3.3 Screening for Functional Aptazymes in Mammalian Cells
	4 Notes
	References
Chapter 15: Aptazyme-Based Riboswitches and Logic Gates in Mammalian Cells
	1 Introduction
	2 Materials
		2.1 Cell Culture Reagents and Cell  Line
		2.2 Equipment
		2.3 Plasmids and Primers
		2.4 Other Reagents and Supplies
	3 Methods
		3.1 Design and Construction of ON and OFF Ribozyme Controls
		3.2 Design and Construction of Aptazyme Library
		3.3 Transfection of Aptazyme Library into HEK 293 Cells (See Note 3)
		3.4 EGFP Assay
		3.5 Sequence Analysis
		3.6 Construction of Logic Gates
	4 Notes
	References
Chapter 16: Folding RNA-Protein Complex into Designed Nanostructures
	1 Introduction
	2 Materials
		2.1 RNA Synthesis
		2.2 Protein Synthesis
		2.3 EMSA
		2.4 High-Speed Atomic Force Microscopy (HS-AFM)
	3 Methods
		3.1 RNA Synthesis
		3.2 Protein Synthesis
		3.3 Electrophoretic Mobility Shift Assay (EMSA)
		3.4 High-Speed Atomic Force Microscopy (HS-AFM)
			3.4.1 Preparation of Mica Surface
			3.4.2 Sample Preparation for AFM Imaging
			3.4.3 AFM Imaging of the Samples
	4 Notes
	References
Chapter 17: An Effective Method for Specific Gene Silencing in Escherichia coli Using Artificial Small RNA
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Bacterial Cell Culture
		2.3 Design and Preparation of Antisense Sequences to Silence Specific Gene Expression
		2.4 Construction of afsRNA Expression Plasmids
		2.5 Evaluation of the Knockdown Efficiency of afsRNA
	3 Methods
		3.1 Design and Preparation of Antisense Sequences for Embedding in an RNA Scaffold
			3.1.1 Consideration of Accessible Regions on Target mRNA
			3.1.2 Synthesis of Oligonucleotides and Annealing
			3.1.3 Phosphorylation of Annealed Products
		3.2 Construction of afsRNA Expression Plasmids
			3.2.1 Preparation of Linearized and Dephosphorylated Vector DNA
			3.2.2 Ligation and Transformation
			3.2.3 Preparation of afsRNA Expression Plasmid
		3.3 Evaluation of the Knockdown Efficiency of Designed afsRNA
			3.3.1 Preparation of afsRNA-Expressing E. coli
			3.3.2 Total RNA Preparation
			3.3.3 DNase Treatment
			3.3.4 cDNA Synthesis
			3.3.5 qPCR Analysis of Gene Expression Suppression by afsRNAs
	4 Notes
	References
Chapter 18: Expression and Purification of tRNA/pre-miRNA-Based Recombinant Noncoding RNAs
	1 Introduction
	2 Materials
		2.1 Cloning, Bacterial Transformation and Culture
			2.1.1 Laboratory Equipment
			2.1.2 Bacterial Culture and Transformation
		2.2 RNA Extraction and Denaturing Urea Polyacrylamide Gel Electrophoresis (PAGE)
			2.2.1 Laboratory Equipment
			2.2.2 RNA Extraction and Denaturing Urea  PAGE
		2.3 RNA Purification
			2.3.1 Laboratory Equipment
			2.3.2 Solutions
		2.4 RNA Purity Analyses
			2.4.1 Laboratory Equipment
			2.4.2 Solutions and Reagents
	3 Methods
		3.1 Design and Construction of BERA/sRNA-Expressing Plasmid
			3.1.1 Design of Target BERA/sRNA and Corresponding Cloning Primers
			3.1.2 PCR Amplification of Target Insert
			3.1.3 Vector Preparation, DNA Product Isolation, and Ligation
			3.1.4 Transformation
			3.1.5 Plasmid Amplification, Mini Preparation, and Sequence Verification
		3.2 Fermentation Production of Target BERA/sRNA
			3.2.1 Small-Scale Expression of BERA/sRNAs
			3.2.2 Large-Scale Expression of BERA/sRNAs
			3.2.3 Isolation of Total Bacterial RNA
			3.2.4 Verification of Target BERA/sRNA Expression
		3.3 Purification of Target BERA/sRNA
			3.3.1 Anion Exchange FPLC Purification
			3.3.2 Desalting and Concentration
		3.4 Analysis of RNA Purity
			3.4.1 Semiquantitative Analysis by Urea-PAGE Analysis
			3.4.2 Quantitative Analysis by  HPLC
			3.4.3 Determination of Endotoxin Level
	4 Notes
	References
Chapter 19: Synthetic Biology Medicine and Bacteria-Based Cancer Therapeutics
	1 Introduction
	2 Materials
		2.1 Bacterial Culture
		2.2 Cell Culture
		2.3 Oral Administration of Bacteria to Mice
		2.4 Intravenous Administration of Bacteria to Mice
	3 Methodology
		3.1 In Vitro Transkingdom Gene Silencing
			3.1.1 Preparation of E. coli
				Inducible tkRNAi Vectors
				Constitutive tkRNAi Vectors
			3.1.2 Preparation of Attenuated S. typhimurium (See Note 3)
				Constitutive tkRNAi Vectors
			3.1.3 Bacterial Infection and Assessment of Target Gene Silencing
				Inducible tkRNAi Vectors
				Constitutive tkRNAi Vectors
		3.2 In-vivo tkRNAi
			3.2.1 Preparation of E. coli
			3.2.2 Oral Treatment of Normal Mice
			3.2.3 Intravenous Treatment of Nude Mice Bearing Colon Cancer Xenografts
			3.2.4 Assessment of Target Gene Knockdown
	4 Notes
	References
Index