Verfügbarkeit: Handbook of RNA biochemistry

Handbook of RNA biochemistry: 2

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Bibliographische Detailangaben
Weitere Verfasser:	Hartmann, Roland K. 1956- (HerausgeberIn)
Format:	Buch
Sprache:	English
Veröffentlicht:	Weinheim WILEY-VCH 2014
Ausgabe:	2., completely rev. and enl. ed.
Schlagworte:	RNS Aufsatzsammlung
Online-Zugang:	Inhaltsverzeichnis
Beschreibung:	Literaturangaben
Beschreibung:	XLII S., S. 547 - 1314 graph. Darst.
ISBN:	9783527327645 9783527647064

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Datensatz im Suchindex

_version_	1804152109973635072
adam_text	Contents to Volume 2 Preface XXIII List of Contributors XXV Part HI RNA Genomics & Bioinformatics, Global Approaches 547 26 Secondary Structure Prediction 549 Gerhard Steger 26.1 Introduction 549 26.2 Thermodynamics 550 26.3 Fonnál Background 552 26.4 mfeldandUBíÄFoId 555 26.4.1 Input to the mf old Server 556 26.4.1.1 Sequence Name 556 26.4.1.2 Sequence 556 VIII Contents 26.4.1.3 Constraints 556 26.4.1.4 Further Parameters 558 26.4.1.5 Immediate versus Batch Jobs 561 26.4.2 Output from the infold Server 561 26.4.2.1 Energy Dot Plot 561 26.4.2.2 Extra Files 563 26.4.2.3 Download All Foldings 563 26.4.2.4 View ss-Count Information 564 26.4.2.5 View Individual Structures 564 26.4.2.6 Dot Plot Folding Comparisons 565 26.5 RNAfold 565 26.5.1 Input to the KNAfold Server 566 26.5.1.1 Sequence and Constraints 566 26.5.1.2 Further Parameters 567 26.5.1.3 Immediate versus Batch Jobs 568 26.5.2 Output from the RNAfold Server 570 26.5.2.1 Text Output of Secondary Structure 570 26.5.2.2 Probability Dot Plot 570 26.5.2.3 Graphical Output of Secondary Structure 570 26.5.2.4 Mountain Plot 571 26.6 Troubleshooting 571 Acknowledgment 573 References 573 27 RNA Secondary Structure Analysis Using Abstract Shapes 579 Robert Giegerich and Björn Voß 27.1 Introduction to Abstract Shape Analysis 579 27.1.1 Looking Deeper into the RNA Folding Space 579 Xl .X.I Overview of Functions of Abstract Shape Analysis 580 27.1.3 Definition of Shape Abstraction 580 27.1.3.1 Shapes 580 27.1.3.2 Shape Abstraction Function 581 27.1.3.3 Shape Representative Structures (shreps) 582 27.1.3.4 Levels of Abstraction 581 27.1.3.5 Shape Probabilities 582 27-1.3.6 Consensus Shape 582 27.1.4 General Caveats when Working with Abstract Shapes 582 27.1.5 Applications of Abstract Shape Analysis 583 27.2 Protocol 1: Computing Shape Representative Structures 584 27.2.1 Useful Parameters for RNAshapes 585 273 Protocol 2: Probabilistic Shape Analysis 585 27.3.1 Useful Parameters 587 Contents I IX 27.4 Protocol 3: Comparative Shape Analysis from Aligned Sequences 587 27 A.I Useful Parameters for RNAlishapes 588 27.5 Protocol 4: Comparative Shape Analysis from Unaligned Sequences 588 27.5.1 Useful Parameters for RNAshapes 592 27.6 RNAshapes Parameter Overview 592 27.7 RNAlishapes Parameter Overview 593 References 594 28 Screening Genome Sequences for known RNA Genes or Motifs 595 Daniel Gautheret 28.1 Introduction 595 28.2 Choosing the Right Search Program 596 28.3 Overview of the RNA Search Procedure 597 28.4 Assessing Search Specificity 598 28.5 A Test Case: Looking for Homologs of a Bacterial sRNA 600 28.5.1 Building a First Training Set with BLASTN 600 28.5.2 Alignment and Structure Prediction 602 28.5.3 Searching with HMMER 604 28.5.4 Searching with RNAMOTIF 606 28.5.5 Searching with ERPIN 609 28.5.6 Searching with INFERNAL 614 28.6 Conclusion 615 28.7 Supplemental Data 625 28.8 Program Versions and Download Sites 616 Acknowledgments 626 References 626 29 Homology Search for Small Structured Non-coding RNAs 62 9 Manja Marz, Stefanie Wehner, and Peter F. Stadler 29.1 Introduction 629 29.2 Materials 629 29.2.1 Sequence Data 629 29.2.2 Web Services 620 29.2.3 Web Service-Independent Software 622 29.3 Protocol: mascRNAs 621 29.3.1 The Seed 622 29.3.2 Low-Hanging Fruits: Initial BLAST Search 623 29.3.3 Initial Secondary Structure Model 624 29.3.4 Drilling Deep - Structure-Based Searches 625 29.4 Concluding Remarks 629 Acknowledgments 630 References 630 X Contents 30 Predict RNA 2D and 3D Structure over the Internet Using MC-Tools 633 Stephen Leong Koan, Jonathan Roy, Marc Parìsien, and François Major 30.1 Introduction 633 30.2 Materials 634 30.2.1 Equipment 634 30.2.2 Data 634 30.3 MC-Tools 635 30.3.1 MC-Fold 635 30.3.2 MC-Cons 636 30.3.3 MC-Sym 636 30.4 Troubleshooting 663 Acknowledgments 663 References 663 31 S2S-Assemble2: a Semi-Automatic Bioinformatics Framework to Study and Model RNA 3D Architectures 667 Fabrice Jossinet and Eric Westhof 31.1 Introduction 667 31.2 S2S: an Interactive RNA Alignment Viewer and Editor 668 31.3 Assemble2: an Interactive RNA 3D Modeler 671 31.4 The Semi-Automatic Architecture of S2S and Assemble2 672 31.5 Installation of S2S andAssemble2 673 References 685 32 Molecular Dynamics Simulations of RNA Systems 687 Pascal Auffinger 32.1 Introduction 687 32.2 MD Methods 689 32.3 Simulation Setups 689 32.3.1 Selecting an Appropriate Starting Structure 689 32.3.1.1 Model-Built Structures 689 32.3.1.2 Х -Ray and Neutron Diffraction Structures 689 32.3.1.3 Cryo-Electron Microscopy (Cryo-EM) Structures 690 32.3.1.4 NMR Structures 690 32.3.2 Checking the Starting Structure 690 32.3.2.1 Conformational Checks 690 32.3.2.2 Rare Non-covalent Interactions 691 32.3.2.3 Protonation Issues 692 32.3.2.4 Solvent 692 32.3.3 Adding Hydrogen Atoms 693 323 A Choosing the Environment (Crystal, Liquid) and Ion Types 693 32.3.5 Setting the Box Size and Placing the Ions and Water 693 32.3.5.1 Box Size 693 Contents XI 32.3.5.2 Monovalent Ions 693 32.3.5.3 Divalent Ions 694 32.3.5.4 Minimal Salt Conditions 694 32.3.5.5 Water Molecules 694 32.3.5.6 Building Initial Solute and Solvent Configurations 694 32.3.6 Choosing the Program and Force Field 695 32.3.6.1 Programs 695 32.3.6.2 Force Fields 695 32.3.6.3 Parameterization of Modified Nucleotides, Ligands, and Ions 696 32.3.6.4 Clustering Artifacts and Ion Parameters 696 32.3.6.5 Water Models 696 32.3.7 Treatment of Electrostatic Interactions 697 32.3.8 Other Simulation Parameters 697 32.3.8.1 Thermodynamic Ensemble 697 32.3.8.2 Temperature and Pressure 698 32.3.8.3 Shake, Time Steps, and Update of the Non-bonded Pair List 698 32.3.8.4 The Flying Ice Cube Problem 698 32.3.9 Equilibration 699 32.3.10 Sampling 699 32.3.10.1 How Long Should a Simulation Be? 699 32.3.10.2 When to Stop a Simulation 700 32.3.10.3 Multiple Molecular Dynamics (MMD) Simulations 701 32.3.10.4 Simulations of Large Systems 701 32.4 Analysis 702 32.4.1 Evaluating the Quality of the Trajectories 701 32.4.1.1 Consistency Checks 702 32.4.1.2 Comparison with Experimental Data 702 32.4.1.3 Visualization 702 32 АЛ Л Validation through Statistical Survey of Structural Databases 703 32.4.2 Convergence Issues 703 32.4.3 Conformational Parameters 703 32.4.4 Data Analysis 704 32 АЛЛ Clustering 704 32.4.4.2 Analysis Packages 704 32.4.4.3 Solvent Analysis 704 32.5 Perspectives 704 Acknowledgments 705 References 705 33 identification and Characterization of Small Non-coding RNAs in Bacteria 719 Dimitri Podkaminski, Marie Bouvier, and Jörg Vogel 33.1 Introduction 719 33.2 Expression-Based Discovery of sRNAs 720 ХИ I Contents 33.2.1 Microarray 720 33.2.2 High-Throughput Sequencing and RNA-Seq 721 33.2.3 Hfq Coimmunoprecipitation 724 33.3 Expression-Independent Searches 726 33.3.1 Biocomputational Approaches 726 33.3.2 Genomic SELEX 728 33.4 Deciphering the Biological Role of an s RNA 728 33.4.1 sRNA Expression Profile 729 33.4.2 sRNA Deletion 729 33.4.3 s RNA Overexpression 731 33.4.4 sRNA Pulse Expression Combined with Transcriptome Analysis 733 33.4.5 sRNA Libraries 734 33.4.6 Finding sRNA-Associated Proteins 735 33.4.7 Biocomputational Approaches to Find Targets 736 33.5 Experimental Target Validation 737 33.5.1 Reporter Gene Fusions and s RNA Chimera 738 33.5.2 In vitro RNA-RNA Footprinting 739 33.5.3 In vitro Characterization of sRNA Function 741 33.6 Conclusions 742 Acknowledgments 776 References 776 34 The Identification of Bacterial Non-coding RNAs through Complementary Approaches 787 Björn Voß and Wolfgang R. Hess 34.1 Introduction 787 34.2 Computational Prediction 787 34.2.1 Workflow 788 34.2.2 Results and Interpretation 789 34.2.3 Alternative Approaches 790 34.2.4 Troubleshooting 791 34.2.4.1 Choice of Genomes 791 34.2.4.2 Short mRNAs and Dual-Function RNAs 794 34.3 Experimental Approaches for High-Throughput RNomics in Bacteria 794 34.3.1 Microarray Analysis 794 34.3.1.1 Considerations for the Design of Tiling Microarrays 795 34.3.1.2 Considerations for the Design of Expression Microarrays 796 34.3.1.3 Direct Labeling of RNA for Microarray Hybridization 796 34.4 Troubleshooting 799 Acknowledgments 799 References 800 Contents XIII 35 Experimentai RNomics, a Global Approach to Identify Non-coding RNAs in Model Organisms, and RNPomics to Analyze the Non-coding RN Ρ Transcriptome 801 Mathieu Rederstorffand Alexander Hüttenhofer 35.1 Introduction 801 35.2 Computational Analysis of ncRNA Sequences 811 35.3 Notes 812 35.4 Computational Analysis of ncRN A Sequences 816 35.5 Notes 826 Acknowledgments 817 References 817 36 Computational Methods for Gene Expression Profiling Using Next-Generation Sequencing (RNA-Seq) 821 John C. Castle 36.1 Introduction 821 36.2 Procedure Overview 822 36.2.1 Understand the Experiment and the Molecular Biology Protocol 823 36.2.1.1 Library Generation 823 36.2.1.2 Sequencing 825 36.2.2 Align Reads 826 36.2.3 Associate Reads with Transcripts 827 36.2.4 Determine Expression and Uncertainty 828 36.2.5 Normalization 828 36.2.6 Output and Viewing 828 36.2.7 Troubleshooting 829 36.2.8 The Future Is Bright! 830 36.3 Protocols: Useful Algorithms, Formats, and Tools 830 References 830 37 Characterization and Prediction of miRNA Targets 833 Jean Hausser and Mihaela Zavolán 37.1 Introduction 833 37.2 Description 834 37.2.1 Building a Set of Positives and Negatives ; Obtaining Examples of Functional and Non-functional miRNA Binding Sites 835 37.2.1.1 Comparative genomics 836 37.2.1.2 miRNA perturbation and omics 837 37.2.1.3 Immunoprecipitation of RISC components 838 37.2.1.4 Measuring translation repression directly with polysome profiles 839 37.2.1.5 Which data set should one use for inferring properties that characterize functional miRNA binding sites? 839 37.2.2 Properties of Functional miRNA Binding Sites 840 37.2.2.1 The seed binding criterion 840 37..2.2.2 Evolutionary conservation 841 XIV Contents 37.2.2.3 Stability of the miRNA-mRNA duplex 841 37.2.2.4 Structural accessibility 841 37.2.2.5 Sequence composition 842 37.2.2.6 Spatial effects 842 37.2.3 Combining Properties and Examples into a Predictive Model 843 37.2.3.1 Inferring properties that consistently predict miRNA targeting across data sets 843 37.2.3.2 Training a miRNA target prediction model 846 37.3 Troubleshooting 847 37.3.1 Using miRNA target predictions in an experimental setting 847 37.3.1.1 How accurate are miRNA target predictions? 848 37.3.1.2 Which miRNA target prediction method should I use? 849 37.3.1.3 How many targets does a miRNA have? 850 37.3.1.4 Why does a particular high-confidence predicted target not change in response to miRNA overexpression? 850 37.3.1.5 Transcript χ is a target of miRNA y according to method z, yet it does not have an miRNA y seed match in the 3 UTR 850 37.3.1.6 The list of targets predicted by method χ has a different type of identifiers (Entrez Gene ID/RefSeq ID/Ensembl transcript/ ...) than the list predicted by method y or the list that one obtains in a large-scale validation experiment (e.g., microarray measurement) 851 37.3.2 The Complexity of Gene Regulation and its Impact on Designing Accurate miRNA Target Prediction Methods 851 References 853 38 Barcoded cDNA Libraries for miRNA Profiling by Next-Generation Sequencing 861 Markus Hafner, Neil Renwick, John Pena, Aleksandra Mihailovic, and Thomas Tuschl 38.1 Introduction 861 38.2 Overview of the Method 862 38.3 Troubleshooting 872 Acknowledgments 872 References 872 39 Transcriptome-Wide Identification of Protein Binding Sites on RNA by PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) 877 Jessica I. Hoell, Markus Hafner, Markus Landthaler, Manuel Ascano, Thalia A. Farazi, Greg Wardle, Jejf Nusbaum, Pavol Cekan, Mohsen Khorshid, Lukas Burger, Mihaela Zavolán, and Thomas Tuschl 39.1 Introduction 877 39.2 Troubleshooting 897 Contents XV Acknowledgments 897 References 897 40 Global Analysis of Protein-RNA Interactions with Single-Nucleotide Resolution Using ¡CLIP 899 Julian König, Nicholas]. Me Glincy, andjernej Ule 40.1 Introduction 899 40.2 Procedure 900 40.2.1 Overview 900 40.2.2 Antibody and Library Preparation Quality Control 902 40.2.3 Oligormcleotide Design 903 40.2.4 Troubleshooting 904 Acknowledgments 927 References 917 Part IV RNA Function, RNP Analysis, SELEX, RNAi 919 41 Use of RNA Affinity Matrices for the Isolation of RNA Binding Proteins 921 Markus Englert, Bettina Späth, Steffen Schiffer, Sylvia Rösch, Hildburg Beier, and Anita Marchfelder 41.1 Introduction 921 41.2 Applications 927 41.2.1 Purification of the Nuclear tRNase Z from Wheat Germ 927 41.2.2 Purification of the tRNA-Splicing Ligase from Wheat Germ 930 41.3 Notes 932 References 932 42 Biotin-Based Affinity Purification of RNA- Protein Complexes 935 Marco Preußner, Silke Schreiner, Inna Gríshina, Zsófia Palfi,Jingyi Hui, and Albrecht Bindereif 42.1 Introduction 935 42.2 Materials 937 42.2.1 Biotinylated Probes 937 42.2.2 Affinity Matrices 937 42.2.3 Cell Extracts 938 42.2.4 Buffers and Solutions 938 42.2.5 Additional Materials 939 42.3 Methods 939 42.3.1 Affinity Purification of RNA- Protein Complexes (RNPs) 939 42.3.1.1 Depletion of Total Cell Lysáte from SAg-Binding Material (Preclearing} 940 42.3.1.2 Preblocking Streptavidin Agarose Beads 941 42.3.1.3 Affinity Selection of RNPs for Biochemical Studies 941 XVI I Contents 42.3.1.4 Elution of Affinity- S elected RNPs for Functional Studies Ъу a Displacement Oligonucleotide 945 42.3.2 Affinity Purification of Specific RNA Binding Proteins by Biotinylated RNAs 948 42.33 Depletion of Nuclear Extract with Biotinylated RNA 951 42.4 Troubleshooting 952 42.4.1 Biotinylated 2 OMe RNA Oligonucleotides 952 42.4.2 Extracts and Buffers 952 42.4.3 Optimization of the Experimental Conditions, When Yields Are Low 952 42 A A Optimization of the Experimental Conditions in the Case of High Background 953 References 953 43 Affinity Purification of Spliceosomai and Small Nuclear Ribonucleoprotein Complexes 957 Julia Dannenberg, Patrìzia Fabrizio, Cindy L. Will, and Reinhard Lührmann 43.1 Introduction 957 43.2 Immunoaffinity Purification 958 43.2.1 Generation of Antipeptide Antibodies: Peptide Selection Criteria 958 43.3 RNA Aptamer-Based Affinity Purification 963 43.3.1 Approaches for the Isolation of Native Spliceosomai Complexes 963 Acknowledgments 971 References 972 44 Study of RNA-Protein Interactions and RNA Structure in Ribonucleoprotein Particles (RNPs) 975 Virginie Marchand, Annie Mougin, Agnès Méreau, Isabelle Behm-Ansmant, Yuri Motorin, and Christiane Branlant 44.1 Introduction 975 44.2 Methods 978 44.2.1 RNP Reconstitution 978 44.2.1.1 Equipment, Materials, and Reagents 978 44.2.1.2 RNA Preparation and Renaturation Step 980 44.2.2 EMSA 981 44.2.2.1 EMSA Method 981 44.2.2.2 Supershift Method 983 44.2.2.3 Identification of Proteins Contained in RNP by EMSA Experiments Coupled to a Second Gel Electrophoresis and Western Blot Analysis 984 44.2.3 Purification of RNPs Reconstituted in Complex Cellular Extracts 986 44.2 A Methods for RNP Purification Using Tobramycin-Sepharose or MS2-MBP Affinity Chromatography 987 44.2.4.1 Equipment and Materials Common to the Two Approaches 987 Contents XVII 44.2.4.2 RNP Purification Using Tobramycin-Sepharose 987 44.2.4.3 Formation of RNPs in the Cellular Extract 989 44.2.4.4 Elution of Purified RNPs under Native Conditions 989 44.2.4.5 MS2-MBP Affinity Chromatography 989 44.2.4.6 Elution and Analysis of Purified RNPs 990 44.2.4.7 Analysis of the Purified RNP Protein Content 990 44.2.5 Probing of RNA Structure 991 44.2.5.1 Properties of the Probes Used 991 44.2.5.2 Equipment, Material, and Reagents 993 44.2.5.3 Probing Method 994 44.2.6 UV Crosslinking and Immunoselection 999 44.2.6.1 Equipment, Materials, and Reagents 1000 44.2.6.2 UV-Crosslinking Method 1003 44..3 Commentaries and Pitfalls 1005 44.3.1 RNP Purification and Reconstitution 1005 44.3Л.1 RNA Purification and Renaturation 1005 44.3.1.2 EMSA 1005 44.3.1.3 Tobramydn - S epha rose Affinity Chromatography 1006 44.3.2 Probing Conditions 1006 44.3.2.1 Choice of the Probes Used 1006 44.3.2.2 Ratio of RNA/Probes 1007 44.3.3 UV Crosslinking 1008 44.3.3.1 Photoreactivity of Individual Amino Acids and Nucleotide Bases 1008 44.3.3.2 Labeled Nucleotide in RNA 1008 44.3.4 Immunoprecipitations 1008 44.3.4.1 Efficiency of Immunoadsorbents for Antibody Binding 1008 44.4 Troubleshooting 1008 44.4.1 RNP Purification by Tobramycin-Sepharose or MS2-MBP Affinity Chromatography 1008 44.4.2 RNP Reconstitution 1009 44.4.3 RNA Probing 1009 44.4.4 UV Crosslinking 1009 44.4.5 Immunoprecipitations 1009 Acknowledgments 1010 References 1010 45 Immunopurification of Endogenous RNAs Associated with RNA Binding Proteins in vivo 1017 Minna-Liisa Änkö and Karla M. Neiigebauer 45.1 Introduction 1017 45.2 Description of Methods 1017 45.2.1 Overview 1017 45.2.2 Analysis of Coimmunoprecipitated RNA 1022 45.2.2.1 Microarray Analysis of Immunopurified RNA 1022 XVIII Contents 45.2.2.2 RT-PCR Analysis of ïmmimopurified RNA 1024 45.2.2.3 Next-Generation Sequencing of ímmunopurified RNA 1025 45.3 Troubleshooting 1025 45.3.1 Critical Points and Common Problems 1025 45.3.2 Uncrosslinked or Crosslinked RNA Immunoprecipitation 1026 45.3.3 Microarray Data Analysis 1026 45.4 Conclusions 2027 Acknowledgments 1027 References 2027 46 Protein-RNA Crossiinking in Native Ribo nucleop rotei n Particles 2029 Olexandr Dybkov, Henning Urlaub, and Reinhard Lührmann 46.1 Introduction 1029 46.2 Overall Strategy 1030 46.3 UV Crossiinking 2O3Í 46.4 Identification of UV-Induced Protein-RNA Crossiinking Sites by Primer Extension Analysis 2033 46.5 Identification of Crosslinked Proteins 2037 46.6 Troubleshooting 2040 Acknowledgments 2050 References 1050 47 Sedimentation Analysis of Ribonudeoprotein Complexes 2055 Tanja Rosei, Jan Medenbach, Andrey Damianov, Silke Schreiner, and Albrecht Bindereif 47.1 Introduction 1055 47.2 Glycerol Gradient Centrifugation 2056 47.3 Fractionation of Ribonucleoproteins (RNPs) by Cesium Chloride Density Gradient Centrifugation 1061 References 2 065 48 identification and Characterization of RNA Binding Proteins through Three-Hybrid Analysis 2067 Felicia Scott and David R. Engelke 48Л Introduction 2067 48.2 Basic Strategy of the Method 2068 48.3 Detailed Components 1070 48.3.1 Yeast Reporter Strain 2070 48.3.2 Plasmids 1070 48.3.3 Hybrid RNA 2072 48.3.3.1 Technical Considerations for the Hybrid RNA 1072 48.3.4 Activation Domain FP2 2073 48.3.4.1 Technical Considerations for the Activation Domain of FP2 2074 48.3.5 Positive Controls 2075 48.4 Troubleshooting 2079 Contents XIX 48.5 Additional Applications 1081 48.6 Summary 1082 Acknowledgments 1083 References 1083 49 Experimental Identification of MicroRNA Targets 1087 Michaela Beitzinger and Gunter Meister 49.1 Introduction 1087 49.2 Troubleshooting and Notes 1093 49.3 Buffers and Solutions 1094 References 1095 50 Aptamer Selection against Biological Macromolecules: Proteins and Carbohydrates 1097 Franziska Peter and C. Stefan Voertler 50.1 Introduction 1097 50.2 General Strategy 1098 50.2.1 Choosing a Suitable Target 1100 50.2.1.1 Protein Targets 2100 50.2.1.2 Carbohydrate Targets 1101 50.2.2 Immobilization of the Target 1102 50.2.3 Selection Assays 1103 50.2.4 Design and Preparation of the Library 1103 50.3 Running the In vitro Selection Cycle 1104 50.4 Analysis of the Selection Outcome 1106 50.5 Troubleshooting 1107 Acknowledgments 1131 References 1131 51 In Vitro Selection against Small Targets 1139 Dirk Eulberg, Christian Maasch, Werner G. Purschke, and Sven Klussmann 51.1 Introduction 1139 51.2 Target Immobilization 1142 51.2.1 Covalent Immobilization 1143 51.2.1.1 Epoxy-Activated Matrices 1143 51.2.1.2 NHS-Activated Matrices 1145 51.2.1.3 Pyridyl Disuifide-Activated Matrices 1146 51.2.2 Non-covalent Immobilization 1147 51.3 Nucleic Acid Libraries 1148 51.3.1 Library Design 1148 51.3.2 Starting Pool Preparation 1149 51.4 Enzymatics 2250 51.4.1 Reverse Transcription 2152 51.4.2 Polymerase Chain Reaction 2252 XXI Contents 51.4.3 In Vitro Transcription 1153 51.5 Partitioning 1154 51.6 Binding Assays 1159 51.6.1 Equilibrium Dialysis 1159 51.6.2 Equilibrium Filtration Analysis 1160 51.6.3 I socratic Competitive Affinity Chromatography 1161 References 1162 52 SELEX Strategies to Identify Antisense and Protein Target Sites in RNA or hn RN Ρ Complexes 1165 Martin Lützelberger, Martin R. Jakobsen, andj0rgen Kjems 52.1 Introduction 1165 52.1.1 Applications for Antisense 1166 52.1.2 Selecting Protein Binding Sites 1166 52.2 Construction of the Library 1166 52.2.1 Generation of Random DNA Fragments from Genomic or Plasmid DNA 1168 52.2.2 Preparing RNA Libraries from Plasmid, cDNA, or Genomic DNA 1168 52.3 Identification of Optimal Antisense Annealing Sites in RNAs 1169 52.4 Identification of Natural RNA Substrates for Proteins and Other ligands 1171 52.5 Cloning, Sequencing, and Validating the Selected Inserts 1171 52.6 Troubleshooting 1172 52.6.1 Sonication of Plasmid DNA does not Yield Shorter Fragments 1172 52.6.2 Inefficient Ligation 2272 52.6.3 Inefficient Mme I Digestion 2272 52.6.4 The Amplification of the Unselected Library is Inefficient 2 2 73 52.6.5 The Library Appears to be Non-Random in the Unselected Pool 2 273 52.6.6 The Selected RNAs do not Bind to Native Protein 2 273 References 2282 53 Genomic SELEX 2285 Jennifer L. Boots, Katarzyna Matylla-Kulinska, Marek Zywicki, Bob Zimmermann, and Renée Schroeder 53.1 Introduction 2285 53.2 Description of the Methods 2286 53.2.1 Library Construction 2286 53.2.2 Choice of Bait 2288 53.2.3 SELEX Procedure 2288 53.2.3.1 Transcription of Genomic Library into RNA Library 2290 53.2.3.2 Counter Selection 2290 53.2.3.3 Positive Selection 2290 53.2.3.4 Recovery and Amplification of Selected Sequences 2192 53.2.3.5 Neutral SELEX 2292 Contents XXI 53.2.3.6 Cloning and Sequencing 1194 53.2.4 Troubleshooting 1194 53.3 Evaluation of Obtained Sequences 1194 53.3.1 Computational Analysis of SELEX-Derived Sequences 1194 53.3.1.1 Read Filtering and Cleaning 1196 53.3.1.2 Genome Mapping 1196 53.3.1.3 Assembly and Annotation 1197 53.3.1.4 Enrichment Analysis 1197 53.3.1.5 Benefits of Sequencing the Initial Library 1198 53.3.1.6 Identification of the Binding Motif 1198 53.3.2 В iochemìcal Analysis of the Genomic Aptamers 1199 53.3.2.1 Validation of the RNA- Protein Interaction 1199 53.3.2.2 Expression Analysis of Genomic Aptamers 1199 53.3.2.3 Reconstruction of the Whole-Transcript-Comprising Genomic Aptamer 1200 53.3.2.4 Determining the Function of the RNA-Protein Interaction 1200 53.4 Conclusions and Outlook 1202 Acknowledgments 2202 References 2202 54 ín vivo SELEX Strategies 1207 Thomas A. Cooper 54.1 Introduction 1207 54.2 Procedure Overview 1208 54.2.1 Design of the Randomized Exon Cassette 2210 54.2.2 Design of the Minigene 2212 54.2.3 RT-PCR Amplification 22Ï3 54.2.4 Monitoring for Enrichment of Exon Sequences That Function as Splicing Enhancers 1213 54.2.5 Troubleshooting 1214 Acknowledgments 1218 References 2229 55 Gene Silencing Methods Using Vector-Encoded siRN As or miRNAs 2222 Ying Poi Liu and Ben Berkhout 55.1 Introduction 1222 55.2 Background Information 2222 55.3 Construction of shRNA Vectors 2223 55.4 Construction of miRNA Vectors 222S 55.5 Construction of Extended shRN As and lhRN As 2229 55.6 Production of Lentivirai Vectors Encoding Anti-HIV-1 shRNAs or e-shRNAs 2230 55.6.1 Troubleshooting 2234 References 1235 XXII Contents 56 Using Chemical Modification to Enhance siRNA Performance 1243 Jesper В. Bramsen, Arnold Grünweiler, Roland K. Hartmann, and Jürgen Kjems 56.1 introduction 1243 56.2 Numerous siRNA Designs: What siRNA Architecture to Choose? 1243 56.3 siRNA Tolerance Toward Modification 1244 56.4 Tools for Chemical Modification of siRNAs 1245 56.4.1 siRNA Backbone Modifications 1246 56.4.2 Ribose 2 -OH Substitutions 1248 56.4.3 Alteration of the Ribose Backbone 1251 56.4.4 Base Modifications 1252 56.5 Improving siRNA Potency 2252 56.6 Enhancing siRNA Nuclease Resistance 1253 56.6.1 siRNA Stability and Ribonucleases 1253 56.6.2 Strategies for siRNA Stabilization 2254 56.7 Enhancing siRNA Silencing Duration 1255 56.8 siRNA Immunogenicity 1256 56.8.1 Cellular Response to siRNA 1256 56.8.2 Chemical Modification Can Abrogate siRNA Immunogenicity 2257 56.9 Reducing siRNA Off-Target Effects by Chemical Modification 1258 56.9.1 Off-Target Effects Caused by miRNA-Like Activities 1258 56.9.2 Reducing OfF-Targeting by Chemical Modification of the siRNA Guide Strand Seed Region 1258 56.9.3 Avoiding Passenger Strand Off-Targeting 1259 56.10 Chemical Modifications Can Improve siRNA Pharmacokinetics 1259 56.10.1 Enhancing Cellular Delivery by siRNA Conjugation 1260 56.10.2 Altering Biodistribution by siRNA Conjugation 1261 56.11 Chemical Modification of siRNAs - State of the Art 2261 56.12 A Guide for In vivo Studies 2262 References 2265 Appendix: UV Spectroscopy for the Quantitation of RNA 2279 Index 1283
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spelling	Handbook of RNA biochemistry 2 ed. by Roland K. Hartmann ... 2., completely rev. and enl. ed. Weinheim WILEY-VCH 2014 XLII S., S. 547 - 1314 graph. Darst. txt rdacontent n rdamedia nc rdacarrier Literaturangaben RNS (DE-588)4076759-0 gnd rswk-swf (DE-588)4143413-4 Aufsatzsammlung gnd-content RNS (DE-588)4076759-0 s DE-604 Hartmann, Roland K. 1956- (DE-588)12993643X edt (DE-604)BV019662557 2 Erscheint auch als Online-Ausgabe, EPUB 978-3-527-65054-5 Erscheint auch als Online-Ausgabe, MOBI 978-3-527-65053-8 Erscheint auch als Online-Ausgabe, PDF 978-3-527-65055-2 Digitalisierung UB Regensburg - ADAM Catalogue Enrichment application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027238220&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	Handbook of RNA biochemistry RNS (DE-588)4076759-0 gnd
subject_GND	(DE-588)4076759-0 (DE-588)4143413-4
title	Handbook of RNA biochemistry
title_auth	Handbook of RNA biochemistry
title_exact_search	Handbook of RNA biochemistry
title_full	Handbook of RNA biochemistry 2 ed. by Roland K. Hartmann ...
title_fullStr	Handbook of RNA biochemistry 2 ed. by Roland K. Hartmann ...
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