ADARs

1st Edition, Volume 710 - January 27, 2025
Imprint: Academic Press
Editor: Peter Beal
Language: English
Hardback ISBN:
9 7 8 - 0 - 4 4 3 - 3 1 5 8 4 - 8
eBook ISBN:
9 7 8 - 0 - 4 4 3 - 3 1 5 8 5 - 5

ADARs, Volume 710 in this ongoing series, highlights new advances in the field, with this new volume presenting interesting chapters written by an international board of author… Read more

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ADARs, Volume 710 in this ongoing series, highlights new advances in the field, with this new volume presenting interesting chapters written by an international board of authors. Updates in this new release include Yeast as a discovery tool for hyperactive ADARs, X-ray crystallography of ADAR-RNA complexes, Enrichment capture enhances the ability to detect A-to-I editing, Editing specificity of ADAR isoforms, Novel chemical tools for detection methods of A to I sites, En Masse Evaluation of RNA Guides (EMERGe) for ADARs, Mouse models for defining physiological functions of ADARs, Nanopore sequencing to detect A-to-I editing sites, CellREADR: An ADAR-based RNA sensor, and more.

Additional sections cover Site-directed RNA editing with MS2-ADAR systems, Sequencing and Bioinformatic Tools for Accurate Assessment of A to I editing in Complete Transcriptomes, Aptazyme-directed A to I RNA editing, and Obstacles in quantifying A-to-I editing by Sanger Sequencing.

Title of Book
Cover image
Title page
Table of Contents
Series Page
Copyright
Contributors
Preface
Chapter One: Leveraging Saccharomyces cerevisiae for ADAR research: From high-yield purification to high-throughput screening and therapeutic applications
Abstract
1 Introduction
2 High-yield purification of ADARs from Saccharomyces cerevisiae for advanced structural and functional studies
3 High-throughput screening in yeast promotes ADAR structure-function relationships and substrate preferences
4 Yeast-based screening system for optimizing guide RNA sequences that promote the RNA editing of disease-causing mutations
5 Exogenous expression of heterologous ADARs in yeast
References
Chapter Two: Structural analysis of human ADAR2-RNA complexes by X-ray crystallography
Abstract
1 Introduction
2 Synthesis and purification of RNAs
3 hADAR2 overexpression and purification for crystallization
4 TEV overexpression, purification, and storage
5 Confirmation of the formation of high affinity hADAR-RNA complexes using EMSA
6 RNA duplex formation and confirmation by native polyacrylamide gel electrophoresis prior to crystallization trials
7 Formation of hADAR2:RNA complexes and crystallization trials
8 X-ray diffraction data collection, structure determination and structure refinement
References
Chapter Three: A probe-based capture enrichment method for detection of A-to-I editing in low abundance transcripts
Abstract
1 Introduction
2 Probe design
3 RNA preparation
4 Step-by-step method details
5 High-throughput sequencing and analysis
6 Advantages and limitations of the protocol
7 Conclusions and future perspectives
Acknowledgments
References
Chapter Four: Editing specificity of ADAR isoforms
Abstract
1 Introduction
2 Open challenges and future directions
Acknowledgements
References
Chapter Five: EndoVIA for quantifying A-to-I editing and mapping the subcellular localization of edited transcripts
Abstract
1 Introduction
2 Sample preparation
3 Immunostaining
4 Quantifying overall inosine abundance
5 Detecting subcellular localization of edited tran-scripts
6 Summary
Acknowledgments
References
Chapter Six: En masse evaluation of RNA guides (EMERGe) for ADARs
Abstract
1 Introduction
2 EMERGe screening protocol
3 Analysis of EMERGe screening results
4 Application of winning sequences
5 Limitations and future work
References
Chapter Seven: Mouse models for understanding physiological functions of ADARs
Abstract
1 Introduction
2 ADAR1 knockout models
3 ADAR1 protein deficient models
4 ADAR2 loss of function models
5 ADAR3 knockout models
6 Mouse models with retained protein expression: dissecting the in vivo functions of ADAR1
7 ADAR1 biochemical mutants
8 AGS-associated mutations
9 Genetic rescue of ADAR1 mutant mouse models
10 Insights from the complete absence of A-to-I RNA editing
11 Conclusions
Acknowledgments
References
Chapter Eight: Nanopore sequencing to detect A-to-I editing sites
Abstract
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter Nine: CellREADR: An ADAR-based RNA sensor-actuator device
Abstract
1 Introduction
2 CellREADR as a RNA sensor-actuator device
3 Experimental design
4 Materials and equipment
5 Procedures
References
Chapter Ten: Restoration of G to A mutated transcripts using the MS2-ADAR1 system
Abstract
1 Introduction
2 Methods
3 Construction of the ADAR1/MS2 fusion protein
4 Construction of guide RNA molecules
5 Cell culture
6 Transfection
7 RNA extraction and DNA synthesis
8 Confirmation of sequence restoration
9 Analysis of RNA editing by bulk sequencing of PCR products
10 RNA stability assay
11 Half-life measurement
12 Discussion
13 Conclusion
Acknowledgments
References
Chapter Eleven: Bioinformatic approaches for accurate assessment of A-to-I editing in complete transcriptomes
Abstract
1 Introduction
2 Characterizing the editome
3 Detection of editing sites – general considerations
4 Detection approaches for specific classes of sites
5 Conclusion
References
Chapter Twelve: Aptazyme-directed A-to-I RNA editing
Abstract
1 Background
2 Construct design
3 Aptazyme-directed A-to-I RNA editing in cellula
4 Conclusions
Acknowledgments
References
Chapter Thirteen: Obstacles in quantifying A-to-I RNA editing by Sanger sequencing
Abstract
1 Introduction
2 Method: detection and quantification of A-to-I RNA editing by Sanger sequencing
3 Summary
Acknowledgment
References

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