Z-DNA: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: The Origin of Left-Handed Poly[d(G-C)]
1 Introduction
2 Postdoctoral Experience with Arthur Kornberg at Stanford
3 My Transition from Stanford to Göttingen, Germany
4 Fritz Pohl, a Visionary
5 Birth of Poly[d(G-C)] in Göttingen
6 Salt-Dependent ``R-L Transition´´ of Poly[d(G-C)]
7 Birth of the Max Planck Institute for Biophysical Chemistry (1971)
8 Interim Period: 1972 Up to When Z-DNA Appeared in 1979
9 Z-DNA (Accompanied by B-DNA) Is Revealed and Proliferates (1979-)
10 Unfinished Business
11 Concluding Remarks
References
Chapter 2: Characterization of Z-DNA Using Circular Dichroism
1 Introduction
2 Materials
2.1 DNA Preparation
2.2 Protein Preparation
2.3 CD Spectrometer
2.4 CD Characterization of the B- to Z-DNA Transition Induced by caZαPKZ at Physiological Salt Concentration (for Subheading 3...
2.5 CD Characterization of the B- to Z-DNA Transition Induced by Chemicals (for Subheading 3.2)
3 Methods
3.1 DNA Preparation
3.2 Protein Preparation
3.3 CD Spectroscopy (See Note 2)
3.4 CD Characterization of the B- to Z-DNA Transition Induced by Zα at Physiological Salt Concentration (Fig. 1) (See Note 15)
3.4.1 Wavelength Scan
3.4.2 Time-Course Measurement
3.5 CD Characterization of the B- to Z-DNA Transition Induced by Chemicals
3.5.1 Wavelength Scan
3.5.2 Time-Course Measurement
3.6 CD Characterization of the BZ Junction-Forming DNA (Fig. 3)
3.6.1 Wavelength Scan
3.6.2 Time-Course Measurement
4 Notes
References
Chapter 3: Characterization of Z-DNA by Infrared Spectroscopy
1 Introduction
2 Materials
3 Methods
3.1 Spectral Collection
3.2 Spectral Analysis
4 Notes
References
Chapter 4: Crystallization of Z-DNA in Complex with Chemical and Z-DNA Binding Z-Alpha Protein
1 Introduction
2 Materials
2.1 Preparation of Z-DNA-Forming ODNs
2.2 Bacterial Culture for Expression of hZαADAR1
2.3 Purification of hZαADAR1
2.4 Crystallization
3 Methods
3.1 Preparation of Duplex ODNs (Annealing)
3.2 Bacterial Cell Culture and Expression of hZαADAR1
3.3 Protein Purification (Zα Proteins)
3.4 Crystallization of Z-DNA in Complex with Zα Protein or Chemical Stabilizer
4 Notes
References
Chapter 5: NMR Titration Studies in Z-DNA Dynamics
1 Introduction
2 Materials
2.1 M9 Media
2.2 Expression
2.3 Purification
2.4 Preparation of NMR Experiment
3 Methods
3.1 Expression of 15N-Labeled ZBPs
3.2 Purification of 15N-Labeled ZBPs
3.3 Preparation of a DNA Duplex
3.4 Titration of ZBP into DNA
3.5 Titration of DNA into ZBP
3.6 Analysis of Titration Data
4 Notes
References
Chapter 6: Single-Molecule Methods to Study Z-DNA Mechanics and Dynamics
1 Introduction
2 Materials
2.1 DNA Samples
2.2 Protein Samples
2.3 Sample Chamber
2.4 Buffer Solutions
2.5 Objective-Type TIRF-Based Single-Molecule Fluorescence Detection Setup with Dual Channels
2.6 Magnetic Tweezers Setup Combined with Single-Molecule FRET Setup (See Subheading 2.5)
3 Methods
3.1 DNA Sample Preparation
3.2 Protein Assay Preparation
3.3 Chamber Preparation
3.4 Imaging Buffer
3.5 smFRET Assay with Two-Fragment DNA
3.6 DNA Manipulation with smFRET-MT or Magnetic Tweezers Only
3.7 Assay Examples
4 Notes
References
Chapter 7: BZ Junctions and Its Application as Probe (2AP) to Detect Z-DNA Formation and Its Effector
1 Introduction
2 Materials
2.1 2AP-Labeled ODNs
2.2 B-to-Z Transition Monitoring
2.3 Zα Domain (See Chap. 5 for Details)
3 Methods
3.1 Design of 2AP-Labeled ODNs Containing ZFS
3.2 Preparation of Double-Stranded ODNs (ODN Annealing)
3.3 Purification of dsODNs (Option)
3.4 Purification of Z-Alpha Protein (See Chap. 4 for Details)
3.5 Fluorescent-Based Z-DNA Formation Assay
4 Notes
References
Chapter 8: Oligonucleotide Containing 8-Trifluoromethyl-2′-Deoxyguanosine as a Z-DNA Probe
1 Introduction
2 Materials
2.1 8CF3dG Phosphoramidite Synthesis
2.2 Prepare 8CF3dG Labeled Oligonucleotides
2.3 Prepare CD Samples
2.4 Prepare In Vitro and In-Cell 19F-NMR Samples
3 Methods
3.1 Synthesis of 8CF3dG Phosphoramidite
3.2 Synthesis and Purification of 8CF3dG Labeled Oligonucleotides
3.3 CD Sample Preparation and Measurements
3.4 19F NMR Preparation and Measurements
3.4.1 In Vitro 19F NMR Sample Preparation and Measurement
3.4.2 In-Cell 19F NMR Sample Preparation and Measurement
3.5 CD Analysis of 8CF3dG Modified Oligonucleotide
3.6 In Vitro 19F NMR Study of 8CF3dG Modified Oligonucleotide
3.7 In-Cell 19F NMR Study of 8CF3dG Modified Oligonucleotide
4 Notes
References
Chapter 9: Chiroptical Properties of Z-DNA Using Ionic Porphyrins and Metalloporphyrins
1 Introduction
2 Materials
3 Methods
3.1 Interaction with Cationic Porphyrins (ZnT4, H2T4, NiT4)
3.2 Z-DNA/NiTPPS System as Supramolecular Device
3.3 Short Z-DNA Sequences
3.4 BZB Sequences
3.5 Spermine Porphyrin Conjugate (ZnTCPPSpm4)
4 Notes
References
Chapter 10: Construction of a Z-DNA-Specific Recombinant Nuclease Zαα-FOK for Conformation Studies
1 Introduction
2 Materials
2.1 Expression of Zαα-FOK Protein
2.2 Purification of Zαα-FOK Protein
2.3 Z-DNA Cleavage Assay
2.4 Cell-Free Protein Expression of Zαα-FOK.
3 Methods
3.1 Zαα-FOK Expression Vector
3.2 Expression of Zαα-FOK Nuclease
3.3 Purification of Zαα-FOK Nuclease
3.3.1 Cell Lysis
3.3.2 Ni-Affinity Chromatography
3.3.3 Size Exclusion Chromatography
3.3.4 Quality and Quantity Check of Zαα-FOK Nuclease
3.4 In Vitro Z-DNA Cleavage Assay
3.5 Cell-Free Protein Expression of Zαα-FOK Nuclease
4 Notes
References
Chapter 11: Human Heme Oxygenase-1 Promoter Activity Is Mediated by Z-DNA Formation
1 Introduction
2 Materials
2.1 Transfection
2.2 Z-Probe Expression Plasmids
2.3 Chromatin Immunoprecipitation
2.4 Real-Time PCR
3 Methods
3.1 Cell Culture and Transfection
3.2 Cell Lysate Preparation
3.3 Antibody-Bound Dynabeads Preparation and Chromatin Immunoprecipitation
4 Notes
References
Chapter 12: ChIP-Seq Strategy to Identify Z-DNA-Forming Sequences in the Human Genome
1 Introduction
2 Materials
2.1 Expression of Z-DNA-Binding Domain
2.2 Chromatin Immunoprecipitation
2.3 ChIP-Seq Library Preparation
2.4 ChIP-Seq Data Analysis
3 Methods
3.1 Cell Culture and the Expression of Zaa
3.2 Chromatin Immunoprecipitation
3.3 ChIP-Seq Library Preparation and Sequencing
3.4 ChIP-Seq Data Analysis
4 Notes
References
Chapter 13: Detection of Z-DNA Structures in Supercoiled Genome
1 Introduction
2 Materials
2.1 Purification of High Molecular Weight DNA
2.2 Converting Linear Genomic DNA into Supercoiled DNA
2.3 Enrichment of DNA Fragments Surrounding Single-Stranded DNA in Supercoiled Genome
2.4 Common Reagents
2.5 Buffers
2.6 Equipment
2.7 Kits
3 Methods
3.1 Purification of High Molecular Weight DNA
3.2 Converting Linear Genomic DNA into Supercoiled DNA
3.3 Enrichment of DNA Fragments Surrounding Single-Stranded DNA in Supercoiled Genome
3.4 Identification of Z-DNA Structures
4 Notes
References
Chapter 14: Thermogenomic Analysis of Left-Handed Z-DNA Propensities in Genomes
1 Introduction
1.1 Background
1.2 Energetics of Z-DNA Formation
1.3 Statistical Mechanics of the Zipper Model for the B-Z Transition
1.4 ZHUNT: A Computational Approach to Mapping Z-DNA in Genomes
1.5 Validating ZHUNT
1.6 Applications of ZHUNT for Genomic and Phylogenomic Analyses
1.7 mZHUNT for Analyses of Z-DNA in Genomes with Methylated Cytosine
2 Materials
3 Methods
4 Notes
4.1 Input File Format
4.2 Possible Errors Running ZHUNT
4.3 Running ZHUNT or mZHUNT on Local Computer
5 Conclusions and Discussion
References
Chapter 15: DeepZ: A Deep Learning Approach for Z-DNA Prediction
1 Introduction
2 The Input Data
3 Data Compression
4 Deep Learning Architectures
5 Train and Test Set
6 Whole-Genome Annotation with Z-DNA Regions
7 DeepZ Model Interpretation
8 Notes
References
Chapter 16: Methods to Study Z-DNA-Induced Genetic Instability
1 Introduction
2 Materials
2.1 Materials for Screening for Z-DNA-Induced DNA Double-Strand Breaks in Yeast Artificial Chromosomes (Exp #1)
2.2 Materials for Detecting Z-DNA-Induced DSBs on Reporter Vectors Recovered from Mammalian Cells (Exp #2)
2.3 Materials for Detecting Z-DNA-Induced Single- and Double-Strand Breaks in Cell-Free Extracts (Exp #3)
3 Methods
3.1 Methods for Screening for Z-DNA-Induced DNA Double-Strand Breaks in Yeast Artificial Chromosomes (Exp #1)
3.1.1 YAC Construction
3.1.2 Z-DNA-Induced Fragility Assay (FOA Selection of URA3 in Yeast Cells)
3.1.3 Transferring YACs to Mutant Yeast Strains Using a Kar-Cross Protocol (Liquid Method) (See Note 5)
3.2 Methods for Detecting Z-DNA-Induced DSBs on Reporter Vectors Recovered from Mammalian Cells (LM-PCR) [Exp #2]
3.3 Methods for Detecting Z-DNA-Induced Single- and Double-Strand Breaks in Cell-Free Extracts (Exp #3)
4 Notes
References
Chapter 17: Single-Molecule Visualization of B-Z Transition in DNA Origami Using High-Speed AFM
1 Introduction
1.1 Design and Construction of Direct Observation System of Rotation in B-Z Transition in the DNA Frame
1.2 Observation of the Rotation in B-Z Transition in the DNA Frame
1.3 Direct Observation of the Flag Rotation During B-Z Transition in the Equilibrium State
2 Materials
2.1 Design and Preparation of DNA Origami
2.2 Preparation of a DNA Origami Frame with B-Z Transition DNA Components
2.3 High-Speed Atomic Force Microscopy (HS-AFM)
3 Methods
3.1 Design and Preparation of a DNA Origami Frame
3.2 Assembly of the DNA Components in the DNA Frame
3.3 High-Speed AFM Imaging of the Behavior of the B-Z DNA Strands in the DNA Frame
4 Notes
References
Chapter 18: Adoption of A-Z Junctions in RNAs by Binding of Zα Domains
1 Introduction
2 Materials
2.1 General Supplies Needed
2.2 Circular Dichroism
2.3 Isothermal Titration Calorimetry
2.4 Analytical Ultracentrifugation
2.5 Nuclear Magnetic Resonance
3 Methods
3.1 Acquiring and Preparing Protein Zα Samples
3.2 Preparation of RNA and RNA/Zα Complexes
3.3 Circular Dichroism for Quantification of Z-Form
3.4 Calculation of Ez Scores from CD Data to Determine Extent of Z-RNA Formation
3.5 Isothermal Titration Calorimetry to Investigate Affinity and Thermodynamics of Binding
3.6 Sedimentation Velocity Analytical Ultracentrifugation of Z-Conformation-Containing RNA/DNAs Bound to Zα
3.7 Nuclear Magnetic Resonance to Monitor Zα-Dependent Switch from A- to Z-Form
4 Notes
References
Chapter 19: Detecting Z-RNA and Z-DNA in Mammalian Cells
1 Introduction
2 Materials
2.1 Culturing Mouse Embryonic Fibroblasts (MEFs) or L929 Cell Line
2.2 Virus Infection or Treatment with CBL0137
2.3 Immunofluorescence Microscopy
3 Methods
3.1 Influenza a Virus Infection
3.2 Immunofluorescence Detection of Z-RNA
4 Notes
References
Chapter 20: Identification of ADAR1 p150 and p110 Associated Edit Sites
1 Introduction
2 Materials
2.1 Exogenous Expression of p150, p110, and p150/p110 in ADAR1 KO Background
2.2 Total RNA Extraction and Preparation of Libraries
2.3 Sequencing, Alignment, Variant Calling, and Determination of ADAR1-Associated Mismatches and Isoform-Selective Edits (Fig....
2.4 Validation of Edit Sites by Amplicon Sequencing
3 Methods
3.1 Exogenous Expression of p150, p110, and p150/p110 in ADAR1 KO Background
3.2 Total RNA Extraction and Preparation of Libraries
3.3 Sequencing, Alignment, Variant Calling, and Determination of ADAR1-Associated Mismatches and Isoform-Selective Edits
3.4 Validation of Edit Sites by Amplicon Sequencing
4 Notes
References
Chapter 21: Z-DNA and Z-RNA: Methods-Past and Future
1 Introduction
2 A Retrospective
3 The Biology of Z-DNA
4 The Zα Family Domain Structure
5 Making a Z-DNA-Binding wHTH from One That Binds B-DNA
6 Well-Characterized Zα Proteins
7 New Approaches to Z-DNA and Z-RNA Biology
8 Final Thoughts
9 What Will We Find in the Future?
10 What Is There to Do?
References
Index