Handbook of RNA biochemistry:
Gespeichert in:
| Format: | Buch |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
Weinheim
Wiley-VCH
2009
|
| Ausgabe: | 1. student ed. |
| Schlagworte: | |
| Online-Zugang: | Inhaltstext Inhaltsverzeichnis |
| Beschreibung: | Literaturangaben |
| Beschreibung: | XLIII, 931 S. Ill., graph. Darst. 24 cm |
| ISBN: | 9783527325344 |
Internformat
MARC
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| 245 | 1 | 0 | |a Handbook of RNA biochemistry |c ed. by Roland Karl Hartmann ... |
| 250 | |a 1. student ed. | ||
| 264 | 1 | |a Weinheim |b Wiley-VCH |c 2009 | |
| 300 | |a XLIII, 931 S. |b Ill., graph. Darst. |c 24 cm | ||
| 336 | |b txt |2 rdacontent | ||
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Datensatz im Suchindex
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Short Contents
Part I RNA Synthesis 1
1.1 Enzymatic RNA Synthesis, Ligation and Modification 3
1.2 Chemical RNA Synthesis 95
Part II Structure Determination 131
11.1 Molecular Biology Methods 133
11.2 Biophysical Methods 385
11.3 Fluorescence and Single Molecule Studies 453
Part III RNA Genomics and Bioinformatics 489
Part IV Analysis of RNA Function 665
IV.l RNA-Protein Interactions in vitro 667
IV.2 RNA-Protein Interactions in vivo 729
IV.3 SELEX 783
Part V RNAi 895
Appendix: UV Spectroscopy for the Quantitation of RNA 910
VI
Contents
Preface XXXI
List of Contributors XXXV
Part I RNA Synthesis 1
1.1 Enzymatic RNA Synthesis, Ligation and Modification 3
1 Enzymatic RNA Synthesis using Bacteriophage T7 RNA Polymerase 3
Heike Gruegelsiepe, Astrid Schon, LeifA. Kirsebom and Roland K. Hartmann
1.1 Introduction 3
1.2 Description of Method - T7 Transcription in vitro 4
1.2.1 Templates 5
1.2.2 Special Demands on the RNA Product 6
1.2.2.1 Homogeneous 5' and 3' Ends, Small RNAs, Functional Groups at the
5' End 6
1.2.2.2 Modified Substrates 7
1.3 Transcription Protocols 8
1.3.1 Transcription with Unmodified Nucleotides 8
1.3.2 Transcription with 2'-Fluoro-modified Pyrimidine Nucleotides 14
1.3.3 Purification 25
1.4 Troubleshooting 17
1.4.1 Low or No Product Yield 17
1.4.2 Side-products and RNA Quality 17
1.5 Rapid Preparation of T7 RNA Polymerase 17
1.5.1 Required Material 28
1.5.2 Procedure 28
1.5.2.1 Cell Growth, Induction and Test for Expression of T7 RNAP 18
1.5.2.2 Purification of T7 RNAP 19
1.5.3 Notes and Troubleshooting 20
Acknowledgement 21
References 21
Contents VII
2 Production of RNAs with Homogeneous 5' and 3' Ends 22
Mario Mori, Esther Lizano, Dagmar K. Willkomm and Roland K. Hartmann
2.1 Introduction 22
2.2 Description of Approach 23
2.2.1 C;s-cleaving Autocatalytic Ribozyme Cassettes 23
2.2.1.1 The 5' Cassette 23
2.2.1.2 The 3' Cassette 23
2.2.1.3 Purification of Released RNA Product and Conversion of End
Groups 26
2.2.2 Trans-cleaving Ribozymes for the Generation of Homogeneous 3'
Ends 26
2.2.3 Further Strategies Toward Homogeneous Ends 29
2.3 Critical Experimental Steps, Changeable Parameters.
Troubleshooting 29
2.3.1 Construction of Cis-cleaving 5' and 3' Cassettes 29
2.3.2 Dephosphorylation Protocols 33
2.3.3 Protocols for RNase P Cleavage 34
2.3.4 Potential Problems 34
References 35
3 RNA Ligation using T4 DNA Ligase 36
MikkoJ. Frilander and Janne J. Turunen
3.1 Introduction 36
3.2 Overview of the RNA Ligation Method using the T4 DNA Ligase 37
3.3 Large-scale Transcription and Purification of RNAs 38
3.4 Generating Homogeneous Acceptor 3' Ends for Ligation 40
3.5 Site-directed Cleavage with RNase H 42
3.6 Dephosphorylation and Phosphorylation of RNAs 43
3.7 RNA Ligation 44
3.8 Troubleshooting 45
3.9 Protocols 46
Acknowledgments 51
References 51
4 T4 RNA Ligase 53
Tina Persson, Dagmar K. Willkomm and Roland K. Hartmann
4.1 Introduction 53
4.2 Mechanism and Substrate Specificity 54
4.2.1 Reaction Mechanism 54
4.2.2 Early Studies 56
4.2.3 Substrate Specificity and Reaction Conditions 57
4.3 Applications of T4 RNA Ligase 58
4.3.1 End-labeling 58
4.3.2 Circularization 59
4.3.3 Intermolecular Ligation of Polynucleotides 59
VIII Contents
4.4 T4 RNA Ligation of Large RNA Molecules 61
4.5 Application Examples and Protocols 64
4.5.1 Production of Full-length tRNAs 64
4.5.2 Specific Protocols 65
4.5.3 General Methods (GM) 69
4.5.4 Chemicals and Enzymes 70
4.5.4.1 Chemical Synthesis and Purification of Oligoribonucleotides 70
4.5.4.2 Chemicals 71
4.5.4.3 Enzymes 72
4.6 Troubleshooting 72
Acknowledgments 72
References 72
5 Co- and Post-Transcriptional Incorporation of Specific Modifications
Including Photoreactive Croups into RNA Molecules 75
Nathan H. Zahler and Michael E. Harris
5.1 Introduction 75
5.1.1 Applications of RNA Modifications 75
5.1.2 Techniques for Incorporation of Modified Nucleotides 77
5.2 Description 79
5.2.1 5'-End Modification by Transcription Priming 79
5.2.2 Chemical Phosphorylation of Nucleosides to Generate
5'-Monophosphate or 5'-Monophosphorothioate Derivatives 80
5.2.3 Attachment of an Arylazide Photo-crosslinking Agent to a 5'-Terminal
Phosphorothioate 82
5.2.4 3'-Addition of an Arylazide Photo-crosslinking Agent 83
5.3 Troubleshooting 84
References 84
6 3-Terminal Attachment of Fluorescent Dyes and Biotin 86
Dagmar K. Willkomm and Roland K. Hartmann
6.1 Introduction 86
6.2 Description of Method 87
6.3 Protocols 88
6.3.1 3' Labeling 88
6.3.1.1 Biotin Attachment [12] 88
6.3.1.2 Fluorescence Labeling [5] 89
6.3.2 Preparatory Procedures: Dephosphorylation of RNA Produced with
3' Hammerheads 89
6.3.3 RNA Downstream Purifications 90
6.3.3.1 Gel Chromatography 90
6.3.3.2 Purification on Denaturing Polyacrylamide Gels 90
6.3.4 Quality Control 91
6.4 Troubleshooting 91
Contents IX
6.4.1 Problems Caused Prior to the Labeling Reaction 91
6.4.2 Problems with the Labeling Reaction Itself 92
6.4.3 Post-labeling Problems 93
References 93
1.2 Chemical RNA Synthesis 95
7 Chemical RNA Synthesis, Purification and Analysis 95
Brian S. Sproat
7.1 Introduction 95
7.2 Description 97
7.2.1 The Solid-phase Synthesis of RNA 97
7.2.1.1 Manual RNA Synthesis 99
7.2.1.2 Automated RNA Synthesis 100
7.2.2 Deprotection 101
7.2.2.1 Deprotection of Base Labile Protecting Groups 101
7.2.2.2 Desilylation of Trityl-off RNA 102
7.2.2.3 Desilylation of Trityl-on RNA 102
7.2.3 Purification 103
7.2.3.1 Anion-exchange HPLC Purification 103
7.2.3.2 Reversed-phase HPLC Purification of Trityl-on RNA 104
7.2.3.3 Detritylation of Trityl-on RNA 105
7.2.3.4 Desalting by HPLC 106
7.2.4 Analysis of the Purified RNA 107
7.3 Troubleshooting 107
References 110
8 Modified RNAs as Tools in RNA Biochemistry 112
Thomas E. Edwards and Snorri Th. Sigurdsson
8.1 Introduction 112
8.1.1 Modification Strategy: The Phosphoramidite Method 113
8.1.2 Modification Strategy: Post-synthetic Labeling 115
8.2 Description of Methods 116
8.2.1 Post-synthetic Modification: The 2'-Amino Approach 116
8.2.1.1 Reaction of 2'-Amino Groups with Succinimidyl Esters 119
8.2.1.2 Reaction of 2'-Amino Groups with Aromatic Isothiocyanates 119
8.2.1.3 Reaction of 2'-Amino Groups with Aliphatic Isocyanates 120
8.3 Experimental Protocols 120
8.3.1 Synthesis of Aromatic Isothiocyanates and Aliphatic Isocyanates 120
8.3.2 Post-synthetic Labeling of 2'-Amino-modified RNA 122
8.3.3 Post-synthetic Labeling of 4-Thiouridine-modified RNA 125
8.3.4 Verification of Label Incorporation 125
8.3.5 Potential Problems and Troubleshooting 326
References 127
X Contents
Part II Structure Determination 131
II.1 Molecular Biology Methods 133
9 Direct Determination of RNA Sequence and Modification by Radiolabeling
Methods 133
OlafCimple and Astrid Schon
9.1 Introduction 133
9.2 Methods 133
9.2.1 Isolation of Pure RNA Species from Biological Material 134
9.2.1.1 Preparation of Size-fractionated RNA 134
9.2.1.2 Isolation of Single Unknown RNA Species Following a Functional
Assay 134
9.2.1.3 Isolation of Single RNA Species with Partially Known Sequence 135
9.2.2 Radioactive Labeling of RNA Termini 137
9.2.2.1 5'Labeling of RNAs 137
'i.l.l.l 3'Labeling of RNAs 138
9.2.3 Sequencing of End-labeled RNA 140
9.2.3.1 Sequencing by Base-specific Enzymatic Hydrolysis of End-labeled
RNA 141
9.2.3.2 Sequencing by Base-specific Chemical Modification and
Cleavage 144
9.2.4 Determination of Modified Nucleotides by Post-labeling Methods 146
9.2.4.1 Analysis of Total Nucleotide Content 146
9.2.4.2 Determination of Position and Identity of Modified Nucleotides 148
9.3 Conclusions and Outlook 149
Acknowledgments 149
References 149
10 Probing RNA Structures with Enzymes and Chemicals In Vitro and
In Vivo 151
Eric Huntzinger, Maria Possedko, Flore Winter, Herve Moine, Chantal
Ehresmann and Pascale Romby
10.1 Introduction 151
10.2 The Probes 153
10.2.1 Enzymes 153
10.2.2 Chemical Probes 153
10.2.3 Lead(II) 155
10.3 Methods 155
10.3.1 Equipment and Reagents 155
10.3.2 RNA Preparation and Renaturation Step 156
10.3.3 Enzymatic and Lead(II)-induced Cleavage Using End-labeled
RNA 157
10.3.4 Chemical Modifications 160
10.3.5 Primer Extension Analysis 161
10.3.6 In Vivo RNA Structure Mapping 163
Contents XI
10.3.6.1 In Vivo DMS Modification 163
10.3.6.2 In Vivo Lead(II)-induced RNA Cleavages 165
10.4 Commentary 166
10.4.1 Critical Parameters 166
10.4.2 In Vivo Mapping 168
10.5 Troubleshooting 168
10.5.1 In Vitro Mapping 168
10.5.2 In Vivo Probing 169
Acknowledgments 169
References 170
11 Study of RNA-Protein Interactions and RNA Structure in Ribonucleoprotein
Particles 172
Virginie Marchand, Annie Mougin, Agnes Mereau and Christiane Branlant
11.1 Introduction 172
11.2 Methods 175
11.2.1 RNP Purification 175
11.2.2 RNP Reconstitution 176
11.2.2.1 Equipment, Materials and Reagents 176
11.2.2.2 RNA Preparation and Renaturation Step 177
11.2.3 EMSA 178
11.2.3.1 EMSA Method 179
11.2.3.2 Supershift Method 180
11.2.3.3 Identification of Proteins Contained in RNP by EMSA Experiments
Coupled to a Second Gel Electrophoresis and Western Blot
Analysis 184
11.2.4 Probing of RNA Structure 185
11.2.4.1 Properties of the Probes Used 185
11.2.4.2 Equipment, Material and Reagents 186
11.2.4.3 Probing Method 187
11.2.5 UV Crosslinking and Immunoselection 195
11.2.5.1 Equipment, Materials and Reagents 195
11.2.5.2 UV Crosslinking Method 196
11.3 Commentaries and Pitfalls 196
11.3.1 RNP Purification and Reconstitution 198
11.3.1.1 RNA Purification and Renaturation 198
11.3.1.2 EMSA 199
11.3.2 Probing Conditions 199
11.3.2.1 Choice of the Probes Used 199
11.3.2.2 Ratio of RNA/Probes 200
11.3.3 UV Crosslinking 200
11.3.3.1 Photoreactivity of Individual Amino Acids and Nucleotide Bases 200
11.3.3.2 Labeled Nucleotide in RNA 201
11.3.4 Immunoprecipitations 201
11.3.4.1 Efficiency of Immunoadsorbents for Antibody Binding 201
XII
Contents
11.4 Troubleshooting 201
11.4.1 RNP Reconstitution 201
11.4.2 RNA Probing 201
11.4.3 UV Crosslinking 202
11.4.4 Immunoprecipitations 202
Acknowledgments 202
References 202
12 Terbium(lll) Footprinting as a Probe of RNA Structure and Metal-binding
Sites 205
Dinari A. Harris and Nils C. Walter
12.1 Introduction 205
12.2 Protocol Description 206
12.2.1 Materials 206
12.3 Application Example 210
12.4 Troubleshooting 212
References 213
13 Pb2+-induced Cleavage of RNA 214
LeifA. Kirsebom andjerzy Ciesiolka
13.1 Introduction 214
13.2 Pb2+ -induced Cleavage to Probe Metal Ion Binding Sites, RNA
Structure and RNA-Ligand Interactions 216
13.2.1 Probing High-affinity Metal Ion Binding Sites 216
13.2.2 Pb2+ -induced Cleavage and RNA Structure 220
13.2.3 Pb2+-induced Cleavage to Study RNA-Ligand Interactions 221
13.3 Protocols for Metal Ion-induced Cleavage of RNA 222
13.4 Troubleshooting 225
13.4.1 No Pb2+-induced Cleavage Detected 225
13.4.2 Complete Degradation of the RNA 225
Acknowledgments 226
References 226
14 In Vivo Determination of RNA Structure by Dimethylsulfate 229
Christina Waldsich and Renee Schroeder
14.1 Introduction 229
14.2 Description of Method 230
14.2.1 Cell Growth and In Vivo DMS Modification 230
14.2.2 RNA Preparation 231
14.2.3 Reverse Transcription 232
14.3 Evidence for Protein-induced Conformational Changes within RNA
In Vivo 233
14.4 Troubleshooting 235
References 237
Contents XIII
15 Probing Structure and Binding Sites on RNA by Fenton Cleavage 238
Cesine Bauer and Christian Berens
15.1 Introduction 238
15.2 Description of Methods 240
15.2.1 Fe2+-mediated Cleavage of Native Group I Intron RNA 240
15.2.2 Fe2+-mediated Tetracycline-directed Hydroxyl Radical Cleavage
Reactions 241
15.3 Comments and Troubleshooting 245
References 247
16 Measuring the Stoichiometry of Magnesium Ions Bound to RNA 250
A. J. Andrews and Carol Fierke
16.1 Introduction 250
16.2 Separation of Free Magnesium from RNA-bound Magnesium 251
16.3 Forced Dialysis is the Preferred Method for Separating Bound and Free
Magnesium Ions 252
16.4 Alternative Methods for Separating Free and Bound Magnesium
Ions 254
16.5 Determining the Concentration of Free Magnesium in the
Flow-through 255
16.6 How to Determine the Concentration of Magnesium Bound to the
RNA and the Number of Binding Sites on the RNA 256
16.7 Conclusion 257
16.8 Troubleshooting 258
References 258
17 Nucleotide Analog Interference Mapping and Suppression: Specific
Applications in Studies of RNA Tertiary Structure, Dynamic Helicase
Mechanism and RNA-Protein Interactions 259
Olga Fedorova, Marc Boudvillain, Jane Kawaoka and Anna Marie Pyle
17.1 Background 259
17.1.1 The Role of Biochemical Methods in Structural Studies 259
17.1.2 NAIM: A Combinatorial Approach for RNA Structure-Function
Analysis 262
17.1.2.1 Description of the Method 262
17.1.2.2 Applications 265
17.1.3 NAIS: A Chemogenetic Tool for Identifying RNA Tertiary Contacts and
Interaction Interfaces 268
17.1.3.1 General Concepts 268
17.1.3.2 Applications: Elucidating Tertiary Contacts in Group I and Group II
Ribozymes 269
17.2 Experimental Protocols for NAIM 271
17.2.1 Nucleoside Analog Thiotriphosphates 271
\1.2.2 Preparation of Transcripts Containing Phosphorothioate Analogs 271
17.2.3 Radioactive Labeling of the RNA Pool 273
XIV Contents
17.2.4 The Selection Step of NAIM: Three Applications for Studies of RNA
Function 273
17.2.4.1 Group II Intron Ribozyme Activity: Selection through
Transesterification 273
17.2.4.2 Reactivity of RNA Helicases: Selection by RNA Unwinding 277
17.2.4.3 RNA-Protein Interactions: A One-pot Reaction for Studying
Transcription Termination 279
17.2.5 Iodine Cleavage of RNA Pools 283
17.2.6 Analysis and Interpretation of NAIM Results 284
17.2.6.1 Quantification of Interference Effects 284
17.3 Experimental Protocols for NAIS 287
17.3.1 Design and Creation of Mutant Constructs 287
17.3.2 Functional Analysis of Mutants for NAIS Experiments 289
17.3.3 The Selection Step for NAIS 289
17.3.4 Data Analysis and Presentation 290
Acknowledgments 291
References 291
18 Nucleotide Analog Interference Mapping: Application to the RNase P
System 294
Simona Cuzic and Roland K. Hartmarm
18.1 Introduction 294
18.1.1 Nucleotide Analog Interference Mapping (NAIM) - The
Approach 294
18.1.2 Critical Aspects of the Method 296
18.1.2.1 Analog Incorporation 296
18.1.2.2 Functional Assays 297
18.1.2.3 Factors Influencing the Outcome of NAIM Studies 297
18.1.3 Interpretation of Results 298
18.1.4 Nucleotide Analog Interference Suppression (NAIS) 300
18.2 NAIM Analysis of Cis-cleaving RNase P RNA-tRNA Conjugates 300
18.2.1 Characterization of a Cis-deaving £. coli RNase P RNA-tRNA
Conjugate 300
18.2.2 Application Example 301
18.2.3 Materials 305
18.2.4 Protocols 306
18.2.5 Data Evaluation 311
18.3 Troubleshooting 313
References 317
19 Identification and Characterization of Metal Ion Binding by Thiophilic Metal
Ion Rescue 319
Eric L Christian
19.1 Introduction 319
19.2 General Considerations of Experimental Conditions 323
19.2.1 Metal Ion Stocks and Conditions 323
Contents XV
19.2.2 Consideration of Buffers and Monovalent Salt 324
19.2.3 Incorporation of Phosphorothioate Analogs 325
19.2.4 Enzyme-Substrate Concentration 327
19.2.5 General Kinetic Methods 328
19.2.6 Measurement of Apparent Metal Ion Affinity 329
19.2.7 Characterization of Metal Ion Binding 333
19.2.8 Further Tests of Metal Ion Cooperativity 336
19.3 Additional Considerations 337
19.3.1 Verification of ferei 337
19.3.2 Contributions to Complexity of Reaction Kinetics 338
19.3.3 Size and Significance of Observed Effects 339
19.4 Conclusion 340
Acknowledgments 341
References 341
20 Identification of Divalent Metal Ion Binding Sites in RNA/DNA-metabolizing
Enzymes by Fe(ll)-mediated Hydroxyl Radical Cleavage 345
Yan-Guo Ren, Niklas Henriksson and Anders Virtanen
20.1 Introduction 345
20.2 Probing Divalent Metal Ion Binding Sites 346
20.2.1 Fe(II)-mediated Hydroxyl Radical Cleavage 346
20.2.2 How to Map Divalent Metal Ion Binding Sites 347
20.2.3 How to Use Aminoglycosides as Functional and Structural
Probes 349
20.3 Protocols 350
20.4 Notes and Troubleshooting 352
References 352
21 Protein-RNA Crosslinking in Native Ribonucleoprotein Particles 354
Henning Urlaub, Klaus Hartmuth and Reinhard Luhrmann
21.1 Introduction 354
21.2 Overall Strategy 354
21.3 UV Crosslinking 355
21.4 Identification of UV-induced Protein-RNA Crosslinking Sites by
Primer Extension Analysis 357
21.5 Identification of Crosslinked Proteins 361
21.6 Troubleshooting 364
21.7 Protocols 367
Acknowledgments 372
References 372
22 Probing RNA Structure by Photoaffinity Crosslinking with 4-Thiouridine and
6-Thioguanosine 374
Michael E. Harris and Eric L. Christian
22.1 Introduction 374
22.2 Description 377
XVI Contents
22.2.1 General Considerations: Reaction Conditions and Concentrations of
Interacting Species 377
22.2.2 Generation and Isolation of Crosslinked RNAs 380
22.2.3 Primer Extension Mapping of Crosslinked Nudeotides 381
22.3 Troubleshooting 382
References 384
11.2 Biophysical Methods 385
23 Structural Analysis of RNA and RNA-Protein Complexes by Small-angle
X-ray Scattering 385
Too Pan and Tobin R. Sosnick
23.1 Introduction 385
23.2 Description of the Method 387
23.2.1 General Requirements 387
23.2.2 An Example for the Application of SAXS 389
23.3 General Information 389
23.4 Question 1: The Oligomerization State of P RNA and the RNase P
Holoenzyme 390
23.5 Question 2: The Overall Shape 392
23.6 Question 3: The Holoenzyme-Substrate Complexes 392
23.7 Troubleshooting 395
23.7.1 Problem 1: Radiation Damage and Aggregation 395
23.7.2 Problem 2: High Scattering Background 395
23.7.3 Problem 3: Scattering Results cannot be Fit to Simple Models 396
23.8 Conclusions/Outlook 396
Acknowledgments 396
References 397
24 Temperature-Gradient Gel Electrophoresis of RNA 398
Detlev Riesner and Gerhard Steger
24.1 Introduction 398
24.2 Method 399
24.2.1 Principle 399
24.2.2 Instruments 400
24.2.3 Handling 400
24.3 Optimization of Experimental Conditions 401
24.3.1 Attribution of Secondary Structures to Transition Curves in
TGGE 401
24.3.2 Pore Size of the Gel Matrix 402
24.3.3 Electric Field 402
24.3.4 Ionic Strength and Urea 402
24.4 Examples 402
24.4.1 Analysis of Different RNA Molecules in a Single TGGE 403
24.4.2 Analysis of Structure Distributions of a Single RNA - Detection of
Specific Structures by Oligonucleotide Labeling 405
Contents XVII
24.4.3 Analysis of Mutants 409
24.4.4 Retardation Gel Electrophoresis in a Temperature Gradient for
Detection of Protein-RNA Complexes 409
24.4.5 Outlook 413
References 424
25 UV Melting, Native Cels and RNA Conformation 415
Andreas Werner
25.1 Monitoring RNA Folding in Solution 415
25.2 Methods 417
25.3 Data Analysis 420
25.4 Energy Calculations and Limitations 422
25.5 RNA Concentration 424
25.6 Salt and pH Dependence 424
25.7 Native Gels 426
References 427
26 Sedimentation Analysis of Ribonucleoprotein Complexes 428
Jan Medenbach, Andrey Damianov, Silke Schreiner and Albrecht Bindereif
26.1 Introduction 428
26.2 Glycerol Gradient Centrifugation 429
26.2.1 Equipment 429
26.2.2 Reagents 429
26.2.3 Method 430
26.2.3.1 Preparation of the Glycerol Gradient 430
26.2.3.2 Sample Preparation and Centrifugation 430
26.2.3.3 Preparation of RNA from Gradient Fractions 431
26.2.3.4 Simultaneous Preparation of RNA and Proteins 431
26.2.3.5 Control Gradient with Sedimentation Markers 432
26.2.3.6 Notes and Troubleshooting 433
26.3 Fractionation of RNPs by Cesium Chloride Density Gradient
Centrifugation 434
26.3.1 Equipment 434
26.3.2 Reagents 434
26.3.3 Method 435
26.3.3.1 Preparation of the Gradient and Ultracentrifugation 435
26.3.3.2 Preparation of RNA from the Gradient Fractions 435
26.3.3.3 Control Gradient for Density Calculation 435
26.3.3.4 Notes and Troubleshooting 435
Acknowledgments 437
References 437
27 Preparation and Handling of RNA Crystals 438
Boris Franqois, Aurelie Lescoute-Phillips, Andreas Werner and Benefit Masquida
27.1 Introduction 438
XVIII Contents
27.2 Design of Short RNA Constructs 439
27.3 RNA Purification 439
27.3.1 HPLC Purification 439
27.3.2 Gel Electrophoresis 440
27.3.3 RNA Recovery 441
27.3.3.1 Elution of the RNA from the Gel 442
27.3.3.2 Concentration and Desalting 441
27.4 Setting Crystal Screens for RNA 442
27A.I Renaturing the RNA 447
27.4.2 Setting-up Crystal Screens 447
27 A3 Forming Complexes with Organic Ligands: The Example of
Aminoglycosides 447
27A A Evaluate Screening Results 449
27.4.5 The Optimization Process 449
27.5 Conclusions 451
References 452
11.3 Fluorescence and Single Molecule Studies 453
28 Fluorescence Labeling of RNA for Single Molecule Studies 453
Filipp Oesterhelt, Ermo Schwe'mberger and Claus Seidel
28.1 Introduction 453
28.2 Fluorescence Resonance Energy Transfer (FRET) 456
28.2.1 Measurement of Distances via FRET 456
28.3 Questions that can be Addressed by Single Molecule
Fluorescence 458
28.3.1 RNA Structure and Dynamics 459
28.3.2 Single Molecule Fluorescence in Cells 460
28.3.2.1 Techniques used for Fluorescent Labeling RNA in Cells 460
28.3.2.2 Intracellular Mobility 462
28.3.3 Single Molecule Detection in Nucleic Acid Analysis 462
28.3.3.1 Fragment Sizing 462
28.3.3.2 Single Molecule Sequencing 462
28.4 Equipment for Single Molecule FRET Measurements 463
28.4.1.1 Excitation of the Fluorophores 463
28.4.1.2 Fluorescence Detection 464
28.4.1.3 Data Analysis 465
28.5 Sample Preparation 466
28.5.1 Fluorophore-Nudeic Acid Interaction 466
28.5.2 RNA Labeling 466
28.5.2.1 Fluorophores for Single Molecule Fluorescence Detection 466
28.5.2.2 Fluorophores used for FRET Experiments 467
28.5.2.3 Attaching Fluorophores to RNA 467
28.5.2.4 Linkers 468
28.5.3 Fluorescence Background 468
Contents XIX
28.5.3.1 Raman Scattered Light 468
28.5.3.2 Cleaning Buffers 468
28.5.3.3 Clean Surfaces 469
28.5.4 Surface Modification 469
28.5.4.1 Coupling Single Molecules to Surfaces 469
28.5.4.2 Surface Passivation 470
28.5.5 Preventing Photodestruction 470
28.6 Troubleshooting 470
28.6.1.1 Orientation Effects 470
28.6.1.2 Dissociation of Molecular Complexes 471
28.6.1.3 Adsorption to the Surface 471
28.6.1.4 Diffusion Limited Observation Times 471
28.6.1.5 Intensity Fluctuations 472
References 472
29 Scanning Force Microscopy and Scanning Force Spectroscopy of RNA 475
Wolfgang Nellen
29.1 Introduction 475
29.2 Questions that could be Addressed by SFM 477
29.3 Statistics 481
29.4 Scanning Force Spectroscopy (SFS) 481
29.5 Questions that may be Addressed by SFS 483
29.6 Protocols 483
29.7 Troubleshooting 485
29.8 Conclusions 486
Acknowledgments 487
References 487
Part III RNA Cenomics and Bioinformatics 489
30 Comparative Analysis of RNA Secondary Structure: 6S RNA 491
James W. Brown andj. Christopher Ellis
30.1 Introduction 491
30.1.1 RNA Secondary Structure 492
30.1.2 Comparative Sequence Analysis 492
30.1.3 Strengths and Weakness of Comparative Analysis 493
30.1.4 Comparison with Other Methods 494
30.2 Description 495
30.2.1 Collecting Sequence Data 495
30.2.2 Thermodynamic Predictions 498
30.2.3 Initial Alignment 500
30.2.4 Terminal Helix (Pla) 502
30.2.5 Subterminal Helix (Plb) 506
30.2.6 Apical Helix (P2a) 506
XX Contents
30.2.7 Subapical Helices (P2b and P2c) 507
30.2.8 Potential Interior Stem-loop (P3) 508
30.2.9 Is There Anything Else? 508
30.2.10 Where To Go From Here 509
30.3 Troubleshooting 510
Acknowledgments 511
References 511
31 Secondary Structure Prediction 513
Gerhard Steger
31.1 Introduction 513
31.2 Thermodynamics 513
31.3 Formal Background 516
31.4 mfold 518
31.4.1 Input to the mfold Server 5i8
31.4.1.1 Sequence Name 518
31.4.1.2 Sequence 518
31.4.1.3 Constraints 519
31.4.1.4 Further Parameters 521
31.4.1.5 Immediate versus Batch Jobs 522
31.4.2 Output from the mfold Server 524
31.4.2.1 Energy Dot Plot 524
31.4.2.2 RNAML (RNA Markup Language) Syntax 524
31.4.2.3 Extra Files 524
31.4.2.4 Download All Foldings 525
31.4.2.5 View ss-count Information 525
31.4.2.6 View Individual Structures 525
31.4.2.7 Dot Plot Folding Comparisons 527
31.5 RNAfold 527
31.5.1 Input to the RNAfold Server 528
31.5.1.1 Sequence and Constraints 528
31.5.1.2 Further Parameters 529
31.5.1.3 Immediate versus Batch Jobs 530
31.5.2 Output from the RNAfold Server 531
31.5.2.1 Probability Dot Plot 531
31.5.2.2 Text Output of Secondary Structure 531
31.5.2.3 Graphical Output of Secondary Structure 531
31.5.2.4 Mountain Plot 533
31.6 Troubleshooting 533
References 534
32 Modeling the Architecture of Structured RNAs within a Modular and
Hierarchical Framework 536
Benolt Masquida and Eric Westhof
32.1 Introduction 536
Contents XXI
32.2 Modeling Large RNA Assemblies 537
32.2.1 The Modeling Process 538
32.2.1.1 Getting the Right Secondary Structure 539
32.2.1.2 Extrusion of the Secondary Structure in 3-D 540
32.2.1.3 Interactive Molecular Modeling 540
32.2.1.4 Refinement of the Model 542
32.3 Conclusions 543
References 544
33 Modeling Large RNA Assemblies using a Reduced Representation 546
Jason A. Mears, Scott M. Stagg and Stephen C. Harvey
33.1 Introduction 546
33.2 Basic Modeling Principles 547
33.2.1 Pseudo-atoms and Reduced Representation 549
33.2.2 Implementing RNA Secondary Structure 550
33.2.3 Protein Components 551
33.2.4 Implementing Tertiary Structural Information 55i
33.2.5 Modeling Protocol 552
33.3 Application of Modeling Large RNA Assemblies 554
33.3.1 Modeling the Ribosome Structure at Low Resolution 554
33.3.2 Modeling Dynamic Assembly of the Ribosome with Reduced
Representation 556
33.4 Conclusion 557
33.5 Troubleshooting 557
References 559
34 Molecular Dynamics Simulations of RNA Systems 560
Pascal Aujfinger and Andrea C. Vaiana
34.1 Introduction 560
34.2 MD Methods 560
34.3 Simulation Setups 562
34.3.1 Choosing the Starting Structure 562
34.3.1.1 Model Built Structures 563
34.3.1.2 X-ray Structures 563
34.3.1.3 NMR Structures 563
34.3.2 Checking the Starting Structure 563
34.3.2.1 Conformational Checks 563
34.3.2.2 Protonation Issues 564
34.3.2.3 Solvent 564
34.3.3 Adding Hydrogen Atoms 564
34.3.4 Choosing the Environment (Crystal, Liquid) and Ions 564
34.3.5 Setting the Box Size and Placing the Ions 565
34.3.5.1 Box Size 565
34.3.5.2 Monovalent Ions 565
34.3.5.3 Divalent Ions 565
XXII Contents
34.3.6 Choosing the Program and Force Field 565
34.3.6.1 Programs 565
34.3.6.2 Force Fields 566
34.3.6.3 Parameterization of Modified Nucleotides and Ligands 566
34.3.6.4 Water Models 567
34.3.7 Treatment of Electrostatic Interactions 567
34.3.8 Other Simulation Parameters 568
34.3.8.1 Thermodynamic Ensemble 568
34.3.8.2 Temperature and Pressure 568
34.3.8.3 Shake, Time Steps and Update of the Non-bonded Pair List 568
34.3.8.4 The Flying Ice Cube Problem 568
34.3.9 Equilibration 569
34.3.10 Sampling 569
34.3.10.1 How Long Should a Simulation Be? 569
34.3.10.2 When to Stop a Simulation 570
34.3.10.3 Multiple MD (MMD) Simulations 570
34.4 Analysis 570
34.4.1 Evaluating the Quality of the Trajectories 570
34.4.1.1 Consistency Checks 571
34.4.1.2 Comparison with Experimental Data 571
34.4.1.3 Visualization 571
34.4.2 Convergence Issues 571
34.4.3 Conformational Parameters 572
34.4.4 Solvent Analysis 572
34.5 Perspectives 572
Acknowledgments 573
References 573
35 Seeking RNA Motifs in Cenomic Sequences 577
Matthieu Legendre and Daniel Gautheret
35.1 Introduction 577
35.2 Choosing the Right Search Software: Limitations and Caveats 578
35.3 Retrieving Programs and Sequence Databases 583
35.4 Organizing RNA Motif Information 581
35.5 Evaluating Search Results 583
35.6 Using the RNAMOTIF Program 585
35.7 Using the ERPIN Program 589
35.8 Troubleshooting 592
35.8.1 RNAMOTIF 592
35.8.1.1 Too Many Solutions 592
35.8.1.2 Program Too Slow 592
35.8.2 ERPIN 592
35.8.2.1 Too Many Solutions 592
35.8.2.2 Program Too Slow 593
Acknowledgments 593
References 593
Contents XXIII
36 Approaches to Identify Novel Non-messenger RNAs in Bacteria and to
Investigate their Biological Functions: RNA Mining 595
Jorg Vogel and E. Gerhart H. Wagner
36.1 Introduction 595
36.2 Searching for Small Untranslated RNAs 597
36.2.1 Introduction 597
36.2.2 Direct Labeling and Direct Cloning 598
36.2.3 Functional Screens 599
36.2.4 Biocomputational Screens 602
36.2.5 Microarray Detection 605
36.2.6 Shotgun Cloning (RNomics) 606
36.2.7 Co-purification with Proteins or Target RNAs 609
36.2.8 Screens for Cis-encoded Antisense RNAs 610
36.3 Conclusions 630
Acknowledgments 62 2
References 611
37 Approaches to Identify Novel Non-messenger RNAs in Bacteria and to
Investigate their Biological Functions: Functional Analysis of Identified
Non-mRNAs 624
E. Gerhart H. Wagner and Jorg Vogel
37.1 Introduction 614
37.2 Approaches for Elucidation of Bacterial sRNA Function 625
37.2.1 Large-scale Screening for Function 625
37.2.2 Preparing for Subsequent Experiments: Strains and Plasmids 625
37.2.3 Experimental Approaches 628
37.2.4 Physiological Phenotypes (Lethality. Growth Defects, etc.) 628
37.2.5 Analyzing sRNA Effects on Specific mRNA Levels by Microarrays 629
37.2.6 Analyzing sRNA Effects by Proteomics 620
37.2.7 Analyzing sRNA Effects by Metabolomics 622
37.2.8 Finding Targets by Reporter Gene Approaches 622
37.2.9 Bioinformatics-aided Approaches 623
37.2.10 Prediction of Regulatory Sequences in the Vicinity of sRNA Gene
Promoters 623
37.2.11 Finding Interacting Sites (Complementarity Antisense) 624
37.3 Additional Methods Towards Functional and Mechanistic
Characterizations 625
37.3.1 Finding sRNA-associated Proteins 625
37.3.2 Regulation of the Target RNA - Use of Reporter Gene Fusions 626
37.3.3 Northern Analyses 627
37.3.4 Analysis of sRNAs - RACE and Primer Extensions 627
37.3.5 Structures of sRNAs and Target RNAs 628
37.4 Conclusions 629
37.5 Protocols 629
Acknowledgments 639
References 640
XXIV I Contents
38 Experimental RNomics: A Global Approach to Identify Non-coding RNAs
in Model Organisms 643
Alexander Huttenhofer
38.1 Introduction 643
38.2 Materials 644
38.2.1 Oligonucleotide Primers 644
38.2.2 Enzymes 644
38.2.3 Buffers 644
38.2.4 Reagents, Kits, Vectors and Bacterial Cells 645
38.3 Protocols for Library Construction and Analysis 645
38.4 Computational Analysis of ncRNA Sequences 652
38.5 Troubleshooting 653
Acknowledgments 653
References 653
39 Large-scale Analysis of mRNA Splice Variants by Microarray 655
Young-Soo Kwon, Hai-Ri Li and Xiang-Dong Fu
39.1 Introduction 655
39.2 Overview of RASL Technology 655
39.3 Description of Methods 657
39.3.1 Preparation of Index Arrays 657
39.3.2 Annotation of Alternative Splicing 658
39.3.3 Target Design 658
39.3.4 Preparation of Target Pool 658
39.3.5 The RASL Assay Protocol 659
39.3.6 PCR Amplification 659
39.3.7 Hybridization on Index Array 660
39.3.8 Data Analysis 661
39.4 Troubleshooting 661
39.4.1 System Limitation and Pitfalls 661
39.4.2 Potential Experimental Problems 662
References 663
IV Analysis of RNA Function 665
IV.l RNA-Protein Interactions in vitro 667
40 Use of RNA Affinity Matrices for the Isolation of RNA-binding
Proteins 667
Steffen Schiffer, Sylvia Rdsch, Bett'ma Spath, Markus Englert, Hildburg Beier and
Anita Marchfelder
40.1 Introduction 667
40.2 Materials 668
40.2.1 CNBr-activated Sepharose 4B Affinity Column 668
40.2.2 NHS-activated HiTrap Columns 669
40.3 Methods 669
Contents XXV
40.3.1 Coupling of tRNAs to CNBr-activated Sepharose 4B 669
40.3.2 Coupling of tRNAs to a 5-ml NHS-activated HiTrap Column 671
40.4 Application 672
40.4.1 Purification of the Nuclear RNase Z from Wheat Germ 672
40.5 Notes 674
References 674
41 Biotin-based Affinity Purification of RNA-Protein Complexes 676
Zsofia Palfi, Jingyi Hui and Albrecht Bindereif
41.1 Introduction 676
41.2 Materials 677
41.2.1 Oligonucleotides 677
41.2.2 Affinity Matrices 678
41.2.3 Cell Extracts 678
41.2.4 Buffers and Solutions 679
41.2.5 Additional Materials 679
41.3 Methods 680
41.3.1 Affinity Purification of RNPs 680
41.3.1.1 Depletion of Total Cell Lysate from SAg-binding Material
(Pre-clearing) 680
41.3.1.2 Pre-blocking SAg Beads 682
41.3.1.3 Affinity Selection of RNPs for Structural Studies 681
41.3.1.4 Affinity Selection of RNPs for Functional Studies by Displacement
Strategy 685
41.3.2 Affinity Purification of Specific RNA-binding Proteins by Biotinylated
RNAs 689
41.4 Troubleshooting 691
41.4.1 Biotinylated 2'OMe RNA Oligonucleotides 691
41.4.2 Extracts and Buffers 691
41.4.3 Optimization of the Experimental Conditions: When Yields are
Low 691
41.4.4 Optimization of the Experimental Conditions: When Non-specific
Background is Too High 692
References 692
42 Immunoaffinity Purification of Spliceosomal and Small Nuclear
Ribonucleoprotein Complexes 694
Cindy L Will, Evgeny M. Makarov, Olga V. Makarova and Reinhard LOhrmann
42.1 Introduction 694
42.2 Generation of Anti-peptide Antibodies: Peptide Selection Criteria 694
42.3 Immunoaffinity Selection of U4/U6.U5 Tri-snRNPs 697
42.4 Immunoaffinity Purification of 17S U2 snRNPs 699
42.5 Approaches for the Isolation of Native, Human Spliceosomal
Complexes 702
42.6 Isolation of Activated Spliceosomes by Immunoaffinity Selection with
Anti-peptide Antibodies against the SKIP Protein 703
XXVI Contents
Acknowledgments 709
References 709
43 Northwestern Techniques for the Identification of RNA-binding Proteins
from cDNA Expression Libraries and the Analysis of RNA-Protein
Interactions 710
Angel Emilio Martinez de Alba, Michela Alexandra Denti and Martin Tabler
43.1 Introduction 710
43.2 Methods 732
43.2.1 Preparation of Probes and Buffers 712
43.2.1.1 Preparation of 32P-labeled RNA Probes 712
43.2.1.2 Preparation of Blocking RNA 712
43.2.1.3 Preparation of the Northwestern Buffer 713
43.2.2 Protocol 1: Northwestern Screening for Identification of RNA-binding
Proteins from cDNA Expression Libraries 713
43.2.2.1 Preparation of the Host Plating Culture 714
43.2.2.2 Plating of the cDNA Phage Expression Library 714
43.2.2.3 Adsorbing Recombinant Proteins to Nitrocellulose Membranes 715
43.2.2.4 Incubation with an RNA Ligand 716
43.2.2.5 Washing of Membranes 717
43.2.2.6 Identification of True Positives 717
43.2.3 Protocol 2: Northwestern Techniques to Detect and Analyze RNA-
Protein Interactions 719
43.2.3.1 Protein Sample Preparation 719
43.2.3.2 Protein Electrophoresis and Transfer 721
43.2.3.3 Incubation of the Membranes with an RNA Probe 722
43.2.3.4 Washing of Membranes and Autoradiography 722
43.3 Troubleshooting 723
43.3.1 Probe Quality 723
43.3.2 Background Signals 724
43.3.3 Signal-to-Background Ratio 724
43.3.4 Protein Conformation 726
43.3.5 Weak Binding Signals 726
43.3.6 False Positives 726
43.3.7 Quality of the cDNA Library 727
43.3.8 Fading Signals 727
43.3.9 Supplementary 727
References 727
IV.2 RNA-Protein Interactions in vivo 729
44 Fluorescent Detection of Nascent Transcripts and RNA-binding Proteins
in Cell Nuclei 729
Jennifer A. Ceiger and Karla M. Neugebauer
44.1 Introduction 729
44.2 Description of the Methods 730
Contents XXVII
44.2.1 Overview 730
44.2.2 Preparation of Fluorescent DNA Probes for In Situ Hybridization 731
44.2.2.1 Method 1: Nick Translation of Plasmid DNA 731
44.2.2.2 Method 2: PCR Amplification and DNase I Digestion 732
44.2.3 Performing Combined Immunocytochemistry and FISH 733
44.2.4 Troubleshooting 735
Acknowledgments 735
References 735
45 Identification and Characterization of RNA-binding Proteins through
Three-hybrid Analysis 737
Felicia Scott and David R. Engelke
45.1 Introduction 737
45.2 Basic Strategy of the Method 738
45.3 Detailed Components 739
45.3.1 Yeast Reporter Strain 740
45.3.2 Plasmids 740
45.3.3 Hybrid RNA 740
45.3.3.1 Technical Considerations for the Hybrid RNA 742
45.3.4 Activation Domain FP2 743
45.3.4.1 Technical Considerations for the Activation Domain FP2 743
45.3.5 Positive Controls 744
45.4 Protocols 745
45.4.1 Transformation of Yeast 745
45.4.2 Assaying for HIS3 Expression 748
45.4.3 Assaying for /i-Galactosidase Activity 748
45.5 Troubleshooting 749
45.6 Additional Applications 75 i
45.7 Summary 752
Acknowledgments 752
References 752
46 Analysis of Alternative Splicing In Vivo using Minigenes 755
Yesheng Tang, Tatyana Novoyatleva, Natalya Benderska, Shivendra Kishore,
Alphonse Thanaraj and Stefan Stamm
46.1 Introduction 755
46.2 Overview of the Method 755
46.3 Methods 757
46.3.1 Construction of Minigenes 757
46.3.2 Transfection of Cells 757
46.3.3 Analysis 771
46.3.3.1 RT-PCR 771
46.3.3.2 Other Analysis Methods 771
46.3.4 Necessary Controls 773
46.3.5 Advantages and Disadvantages of the Method 773
46.3.6 Related Methods 774
XXVIII Contents
46.4 Troubleshooting 774
46.5 Bioinformatic Resources 775
46.6 Protocols 775
References 780
IV.3 SELEX 783
47 Artificial Selection: Finding Function amongst Randomized Sequences 783
Ico de Zwart, Catherine Lozupone, Rob Knight, Amanda Birmingham,
Mali Illangasekare, Vasant Jadhav, Michal Legiewicz, Irene Majerfeld,
Jeremy Widmann and Michael Yarus
47.1 The SELEX Method 783
47.2 Understanding a Selection 784
47.2.1 Sequence Motif Representation and Abundance 786
47.2.2 The Recovery Efficiency of Different RNAs 787
47.2.3 Stringency 789
47.2.4 Amplification and Transcription Biases 789
47.3 Isolation of RNAs that Bind Small Molecules 790
47.3.1 Stringency and KD 792
47.3.2 Selection for Multiple Targets in One Column 793
47.3.3 Characterizing Motif Activity 793
47.4 Techniques for Selecting Ribozymes 794
47.4.1 Making the RNA a Substrate for the Reaction 795
47.4.2 The Inherent Reactivity of RNA 795
47.4.3 Selecting Active RNAs 797
47.4.4 Negative Selections 798
47.4.5 Stringency 799
47.4.6 Analysis of the Product 799
47.4.7 Determining the Scope of the Reaction 799
47.5 Sequence Analysis 800
47.5.1 Identifying Related Sequences 800
47.5.2 Predicting Structure 802
47.5.3 Chemical and Enzymatic Mapping 803
47.5.4 Finding Minimal Requirements 804
47.5.5 Three-dimensional Structural Modeling 804
Acknowledgments 805
References 805
48 Aptamer Selection against Biological Macromolecules: Proteins and
Carbohydrates 807
C. Stefan Vortler and Maria Milovnikova
48.1 Introduction 807
48.2 General Strategy 808
48.2.1 Choosing a Suitable Target 810
48.2.1.1 Protein Targets 810
48.2.1.2 Carbohydrate Targets 811
Contents XXIX
48.2.2 Immobilization of the Target 812
48.2.3 Selection Assays 822
48.2.4 Design and Preparation of the Library 813
48.3 Running the In Vitro Selection Cycle 814
48.4 Analysis of the Selection Outcome 815
48.5 Troubleshooting 816
48.6 Protocols 817
Acknowledgments 836
References 836
49 In Vivo SELEX Strategies 840
Thomas A. Cooper
49.1 Introduction 840
49.2 Procedure Overview 841
49.2.1 Design of the Randomized Exon Cassette 843
49.2.2 Design of the Minigene 844
49.2.3 RT-PCR Amplification 846
49.2.4 Monitoring for Enrichment of Exon Sequences that Function as
Splicing Enhancers 846
49.2.5 Troubleshooting 847
49.3 Protocols 848
Acknowledgments 852
References 852
50 In Vitro Selection against Small Targets 853
Dirk Eulberg, Christian Maasch, Werner C. Purschke and Sven Klussmann
50.1 Introduction 853
50.2 Target Immobilization 856
50.2.1 Covalent Immobilization 857
50.2.1.1 Epoxy-activated Matrices 857
50.2.1.2 NHS-activated Matrices 859
50.2.1.3 Pyridyl Disulfide-activated Matrices 860
50.2.2 Non-covalent Immobilization 861
50.3 Nucleic Acid Libraries 862
50.3.1 Library Design 862
50.3.2 Starting Pool Preparation 863
50.4 Enzymatics 865
50.4.1 Reverse Transcription 865
50.4.2 PCR 866
50.4.3 In Vitro Transcription 867
50.5 Partitioning 868
50.6 Binding Assays 873
50.6.1 Equilibrium Dialysis 873
50.6.2 Equilibrium Filtration Analysis 874
50.6.3 Isocratic Competitive Affinity Chromatography 875
References 877
XXX Contents
51 SELEX Strategies to Identify Antisense and Protein Target Sites in RNA or
Heterogeneous Nuclear Ribonucleoprotein Complexes 878
Martin Lutzelberger, Martin R. Jakobsen and ]0rgen Kjems
51.1 Introduction 878
51.1.1 Applications for Antisense 879
51.1.2 Selecting Protein-binding Sites 879
51.2 Construction of the Library 879
51.2.1 Generation of Random DNA Fragments from Genomic or Plasmid
DNA 881
51.2.2 Preparing RNA Libraries from Plasmid, cDNA or Genomic DNA 881
51.3 Identification of Optimal Antisense Annealing Sites in RNAs 882
51.4 Identification of Natural RNA Substrates for Proteins and Other
Ligands 884
51.5 Cloning, Sequencing and Validating the Selected Inserts 884
51.6 Troubleshooting 885
51.6.1 Sonication of Plasmid DNA does not Yield Shorter Fragments 885
51.6.2 Inefficient Ligation 885
51.6.3 Inefficient Mmel Digestion 885
51.6.4 The Amplification of the Unselected Library is Inefficient 886
51.6.5 The Library Appears to be Non-random in the Unselected Pool 886
51.6.6 The Selected RNAs do not Bind Native Protein 886
51.7 Protocols 886
References 894
V RNAi 895
52 Gene Silencing Methods for Mammalian Cells: Application of Synthetic
Short Interfering RNAs 897
Matthias John, Anke Ceick, Philipp Hadwiger, Hans-Peter Vomlocher and
Olaf Heidenreich
52.1 Introduction 897
52.2 Background Information 898
52.3 Ways to Induce RNAi in Mammalian Cells 900
52.3.1 Important Parameters 901
52.3.1.1 siRNA Design 902
52.3.1.2 Target Site Selection 901
52.3.1.3 Preparation of siRNA Samples 902
52.3.2 Transfection of Mammalian Cells with siRNA 902
52.3.3 Electroporation of Mammalian Cells with siRNA 904
52.3.4 Induction of RNAi by Intracellular siRNA Expression 905
52.4 Troubleshooting 908
References 908
Appendix: UV Spectroscopy for the Quantitation of RNA 910
Index 915 |
| any_adam_object | 1 |
| author_GND | (DE-588)12993643X |
| building | Verbundindex |
| bvnumber | BV035323087 |
| callnumber-first | Q - Science |
| callnumber-label | QP623 |
| callnumber-raw | QP623 |
| callnumber-search | QP623 |
| callnumber-sort | QP 3623 |
| callnumber-subject | QP - Physiology |
| classification_rvk | WD 5355 |
| classification_tum | CHE 860b |
| ctrlnum | (OCoLC)297798697 (DE-599)DNB991653092 |
| dewey-full | 572.88072 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.88072 |
| dewey-search | 572.88072 |
| dewey-sort | 3572.88072 |
| dewey-tens | 570 - Biology |
| discipline | Biologie Chemie |
| edition | 1. student ed. |
| format | Book |
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| genre | (DE-588)4143413-4 Aufsatzsammlung gnd-content |
| genre_facet | Aufsatzsammlung |
| id | DE-604.BV035323087 |
| illustrated | Illustrated |
| indexdate | 2024-07-20T10:03:32Z |
| institution | BVB |
| isbn | 9783527325344 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-017127627 |
| oclc_num | 297798697 |
| open_access_boolean | |
| owner | DE-91G DE-BY-TUM DE-29T DE-526 DE-20 DE-188 |
| owner_facet | DE-91G DE-BY-TUM DE-29T DE-526 DE-20 DE-188 |
| physical | XLIII, 931 S. Ill., graph. Darst. 24 cm |
| publishDate | 2009 |
| publishDateSearch | 2009 |
| publishDateSort | 2009 |
| publisher | Wiley-VCH |
| record_format | marc |
| spelling | Handbook of RNA biochemistry ed. by Roland Karl Hartmann ... 1. student ed. Weinheim Wiley-VCH 2009 XLIII, 931 S. Ill., graph. Darst. 24 cm txt rdacontent n rdamedia nc rdacarrier Literaturangaben RNA 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- Sonstige (DE-588)12993643X oth text/html http://deposit.dnb.de/cgi-bin/dokserv?id=3191747&prov=M&dok_var=1&dok_ext=htm Inhaltstext HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=017127627&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Handbook of RNA biochemistry RNA 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 ed. by Roland Karl Hartmann ... |
| title_fullStr | Handbook of RNA biochemistry ed. by Roland Karl Hartmann ... |
| title_full_unstemmed | Handbook of RNA biochemistry ed. by Roland Karl Hartmann ... |
| title_short | Handbook of RNA biochemistry |
| title_sort | handbook of rna biochemistry |
| topic | RNA RNS (DE-588)4076759-0 gnd |
| topic_facet | RNA RNS Aufsatzsammlung |
| url | http://deposit.dnb.de/cgi-bin/dokserv?id=3191747&prov=M&dok_var=1&dok_ext=htm http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=017127627&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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