Handbook of RNA biochemistry:
Gespeichert in:
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| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
Weinheim
Wiley-VCH
2014
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| Ausgabe: | 2., completely rev. and enl. ed. |
| Schlagworte: | |
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| Beschreibung: | Literaturangaben |
| Beschreibung: | LIV, 1314 S. Ill., graph. Darst. 24 cm |
| ISBN: | 3527327762 9783527327768 9783527647064 |
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| 245 | 1 | 0 | |a Handbook of RNA biochemistry |c ed. by Roland K. Hartmann ... |
| 250 | |a 2., completely rev. and enl. ed. | ||
| 264 | 1 | |a Weinheim |b Wiley-VCH |c 2014 | |
| 300 | |a LIV, 1314 S. |b Ill., graph. Darst. |c 24 cm | ||
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Datensatz im Suchindex
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CONTENTS
PREFACE XXXV
LIST OF CONTRIBUTORS XXXVII
PART I RNA SYNTHESIS AND DETECTION 1
1 ENZYMATIC RNA SYNTHESIS USING BACTERIOPHAGE T7 RNA POLYMERASE 3
MARKUS GDJIRINGER, DOMINIK HELMECKE, KAREN KOHLER, ASTRID
SCHON,
LEIF A. KIRSEBOM, ALBRECHT
BINDEREIF, AND ROLAND K. HARTMANN
1.1 INTRODUCTION 3
1.2 DESCRIPTION OF METHOD - T7 TRANSCRIPTION IN VITRO 4
1.2.1 TEMPLATES 4
1.2.1.1 STRATEGY (I): INSERTION INTO A PLASMID 4
1.2.1.2 STRATEGY (II): DIRECT USE OF TEMPLATES GENERATED BY PCR 5
1.2.1.3 STRATEGY (III): ANNEALING OF A T7 PROMOTER DNA OLIGONUCLEOTIDE
TO A SINGLE-STRANDED TEMPLATE 5
1.2.2 SPECIAL DEMANDS ON THE RNA PRODUCT 5
1.2.2.1 HOMOGENEOUS 5' AND 3' ENDS, SMALL RNAS, FUNCTIONAL GROUPS
AT THE 5' END 5
1.2.2.2 MODIFIED SUBSTRATES 6
1.3 TRANSCRIPTION PROTOCOLS 8
1.3.1 TRANSCRIPTION WITH UNMODIFIED NUCLEOTIDES 9
1.3.2 TRANSCRIPTION WITH 2'-FLUORO-MODIFIED NUCLEOTIDES 16
1.3.3 T7 TRANSCRIPTS WITH 5'-CAP STRUCTURES 17
1.3.4 PURIFICATION 18
1.4 TROUBLESHOOTING 20
1.4.1 LOW OR NO PRODUCT YIELD 20
1.5 RAPID PREPARATION OF T7 RNA POLYMERASE 21
1.5.1 REQUIRED MATERIAL 21
1.5.1.1 MEDIUM 21
1.5.1.2 BUFFERS AND SOLUTIONS 21
1.5.1.3 ELECTROPHORESIS AND CHROMATOGRAPHY 22
1.5.2 PROCEDURE 22
1.5.2.1 CELL GROWTH, INDUCTION, AND TEST FOR EXPRESSION OF T7 RNAP 22
HTTP://D-NB.INFO/1067782060
VI I CONTENTS
1.5.2.2 PURIFICATION OFT7 RNAP 23
1.5.3 NOTES AND TROUBLESHOOTING 24
REFERENCES 25
2 PRODUCTION OF RNAS WITH HOMOGENEOUS 5'-AND 3'-ENDS 29
MARIO MORI AND ROLAND K. HARTMANN
2.1 INTRODUCTION 29
2.2 DESCRIPTION OF APPROACH 30
2.2.1 CIS-CLEAVING AUTOCATALYTIC RIBOZYME CASSETTES 30
2.2.1.1 THE 5'-CASSETTE 30
2.2.1.2 THE 3'-CASSETTE 30
2.2.1.3 PURIFICATION OF RELEASED RNA PRODUCT AND CONVERSION OF END
GROUPS 31
2.2.2 TRANS-CLEAVING RIBOZYMES FOR THE GENERATION OF HOMOGENEOUS
3' ENDS 33
2.2.3 FURTHER STRATEGIES TOWARD HOMOGENEOUS ENDS 35
2.3 CRITICAL EXPERIMENTAL STEPS, CHANGEABLE PARAMETERS,
TROUBLESHOOTING 36
2.3.1 CONSTRUCTION OF CIS-CLEAVING 5'- AND 3'-CASSETTES 36
2.4 PCR PROTOCOLS 37
2.5 POTENTIAL PROBLEMS 42
REFERENCES 42
3 RNA LIGATION 45
JANNE J. TURUNEN, LIUDMILA V. PAVLOVA, MARTIN HENGESBACH, MARK HELM,
SABINE MIILLER, ROLAND K. HARTMANN, AND MIKKO J. FRILANDER
3.1 GENERAL INTRODUCTION 45
3.1.1 T4 POLYNUCLEOTIDE LIGASES 46
3.1.2 REACTION MECHANISM 46
3.1.3 ADVANTAGES OF T4 DNA LIGASE FOR RNA LIGATION 49
3.1.4 CHAPTER STRUCTURE 49
3.2 RNA LIGATION USING T4 DNA LIGASE (T4 DNL) 50
3.2.1 OVERVIEW OF THE RNA LIGATION METHOD USING THE T4 DNA LIGASE
(T4 DNL) 51
3.2.2 LARGE-SCALE TRANSCRIPTION AND PURIFICATION OF RNAS 53
3.2.3 GENERATING HOMOGENEOUS ACCEPTOR 3'-ENDS FOR LIGATION 53
3.2.4 SITE-DIRECTED CLEAVAGE WITH RNASE H 54
3.2.5 DEPHOSPHORYLATION AND PHOSPHORYLATION OF RNAS 56
3.2.6 RNA LIGATION 57
3.2.7 TROUBLESHOOTING 58
3.3 SIMULTANEOUS SPLINT LIGATION OF FIVE RNA FRAGMENTS TO GENERATE
RNAS FOR FRET EXPERIMENTS 66
3.3.1 INTRODUCTION 66
3.3.2 CONSTRUCT DESIGN 68
3.3.3 TROUBLESHOOTING 70
CONTENTS I VII
3.3.3.1 LOW OVERALL LIGATION EFFICIENCY 70
3.3.3.2 UNDESIRED LIGATION BY-PRODUCTS 70
3.3.3.3 RNA DEGRADATION 70
3.4 T4 RNA LIGASE(S) 70
3.4.1 INTRODUCTION 70
3.4.2 MECHANISM AND SUBSTRATE SPECIFICITY 71
3.4.2.1 EARLY STUDIES 71
3.4.2.2 SUBSTRATE SPECIFICITY AND REACTION CONDITIONS 72
3.4.3 APPLICATIONS OF T4 RNA LIGASE 73
3.4.3.1 END-LABELING 73
3.4.3.2 CIRCULARIZATION 75
3.4.3.3 INTERMOLECULAR LIGATION OF POLYNUCLEOTIDES 75
3.4.4 T4 RNA LIGATION OF LARGE RNA MOLECULES 76
3.4.5 APPLICATION EXAMPLES AND PROTOCOLS 79
3.4.5.1 PRODUCTION OF FULL-LENGTH TRNAS 79
3.4.6 TROUBLESHOOTING 84
REFERENCES 84
4 NORTHERN BLOT DETECTION OF SMALL RNAS 89
BENEDIKT M. BECKMANN, ARNOLD GRIINWELLER, AND ROLAND K. HARTMANN
4.1 INTRODUCTION 89
4.1.1 ISOLATION OF RNA 89
4.1.1.1 KITS 90
4.1.1.2 DO IT YOURSELF 90
4.1.1.3 QUALITY CONTROL 90
4.1.2 NATIVE VERSUS DENATURING GELS 90
4.1.3 TRANSFER OF RNA AND FIXATION TO MEMBRANES 91
4.1.4 HYBRIDIZATION WITH A COMPLEMENTARY PROBE 92
4.1.4.1 DESIGN OF DNA/LNA MIXMER PROBES 92
4.1.5 DETECTION OF DIG-LABELED PROBES 95
4.1.6 TROUBLESHOOTING 95
4.1.7 APPLICATION EXAMPLE 96
4.1.8 LIMITATIONS OF THE METHOD 96
4.2 NORTHERN HYBRIDIZATION PROTOCOLS 98
REFERENCES 102
5 RAPID, NON-DENATURING, LARGE-SCALE PURIFICATION OF IN VITRO
TRANSCRIBED
RNA USING WEAK ANION-EXCHANGE CHROMATOGRAPHY 105
LAURA E. EASTON, YOKO SHIBATA, AND PETER J. LUKAVSKY
5.1 INTRODUCTION 105
5.2 MATERIALS 106
5.2.1 CLONING AND PLASMID PURIFICATION 106
5.2.2 IN VITRO
TRANSCRIPTION 106
5.2.3 WEAK ANION-EXCHANGE FPLC 107
VIII
| CONTENTS
5.3 PROTOCOLS FOR PLASMID DESIGN AND PREPARATION, RNA TRANSCRIPTION,
AND WEAK ANION-EXCHANGE PURIFICATION 107
5.4 TROUBLESHOOTING 115
ACKNOWLEDGMENTS 115
REFERENCES 116
6 3'-TERMINAL ATTACHMENT OF FLUORESCENT DYES AND BIOTIN
DAGMAR K. WILLKOMM AND ROLAND K. HARTMANN
6.1 INTRODUCTION 117
6.2 DESCRIPTION OF METHOD 118
6.3 HISTORY OF THE METHOD 118
6.4 TROUBLESHOOTING 124
6.4.1 PROBLEMS CAUSED BEFORE THE LABELING REACTION 124
6.4.1.1 QUALITY OF THE RNA 3'ENDS 124
6.4.1.2 PURITY OF THE RNA TO BE LABELED 124
6.4.2 PROBLEMS WITH THE LABELING REACTION ITSELF 124
6.4.2.1 PH OF REAGENTS 124
6.4.2.2 STABILITY OF REAGENTS 124
6.4.3 POSTLABELING PROBLEMS 125
6.4.3.1 REMOVAL OF LABELING REAGENTS 125
6.4.3.2 LOSS OF RNA MATERIAL DURING DOWNSTREAM PURIFICATION
6.4.3.3 STABILITY OF LABELED RNA 125
ACKNOWLEDGMENT 125
REFERENCES 125
7 CHEMICAL RNA SYNTHESIS, PURIFICATION, AND ANALYSIS 129
BRIAN S. SPROAT
7.1 INTRODUCTION 129
7.2 DESCRIPTION 132
7.2.1 THE SOLID-PHASE SYNTHESIS OF RNA 132
7.2.2 DEPROTECTION 13 6
7.2.3 PURIFICATION 138
7.2.3.1 ANION-EXCHANGE HPLC PURIFICATION 139
7.2.3.2 REVERSED-PHASE HPLC PURIFICATION OF TRITYL-ON RNA 140
7.2.3.3 DETRITYLATION OF TRITYL-ON RNA 142
7.2.3.4 DESALTING BY HPLC 142
7.2.4 ANALYSIS OF THE PURIFIED RNA 143
7.3 TROUBLESHOOTING 144
REFERENCES 147
8 MODIFIED RNAS AS TOOLS IN RNA BIOCHEMISTRY 151
THOMAS E. EDWARDS AND SNORRI
TH. SIGURDSSON
8.1 INTRODUCTION 151
8.1.1 MODIFICATION STRATEGY: THE PHOSPHORAMIDITE METHOD 152
8.1.2 MODIFICATION STRATEGY: POSTSYNTHETIC LABELING 154
117
125
CONTENTS IX
8.2 DESCRIPTION OF METHODS 156
8.2.1 POSTSYNTHETIC MODIFICATION: THE 2'-AMINO APPROACH 156
8.2.2 REACTION OF 2'-AMINO GROUPS WITH SUCCINIMIDYL ESTERS 158
8.2.3 REACTION OF 2'-AMINO GROUPS WITH AROMATIC ISOTHIOCYANATES 158
8.2.4 REACTION OF 2'-AMINO GROUPS WITH ALIPHATIC ISOCYANATES 159
8.3 EXPERIMENTAL PROTOCOLS 159
8.3.1 SYNTHESIS OF AROMATIC ISOTHIOCYANATES AND ALIPHATIC
ISOCYANATES 160
8.3.2 POSTSYNTHETIC LABELING OF 2'-AMINO-MODIFIED RNA 161
8.3.3 POSTSYNTHETIC LABELING OF 4-THIOURIDINE-MODIFIED RNA 164
8.3.4 VERIFICATION OF LABEL INCORPORATION 164
8.3.5 POTENTIAL PROBLEMS AND TROUBLESHOOTING 165
REFERENCES 166
PART II STRUCTURE DETERMINATION 173
9 DIRECT DETERMINATION OF RNA SEQUENCE AND MODIFICATION BY
RADIOLABELING METHODS 175
OLAF GIMPLE AND ASTRID SCHON
9.1 INTRODUCTION 175
9.2 GENERAL METHODS 175
9.3 ISOLATION OF PURE RNA SPECIES FROM BIOLOGICAL MATERIAL 176
9.3.1 PREPARATION OF SIZE-FRACTIONATED RNA 176
9.3.2 ISOLATION OF A SINGLE UNKNOWN RNA SPECIES FOLLOWING A FUNCTIONAL
ASSAY 176
9.3.2.1 SOLUTIONS FOR ELECTROPHORESIS, STAINING, AND ELUTION OF RNAS
FROM
GELS 176
9.3.2.2 TWO-DIMENSIONAL ELECTROPHORESIS OF RNA 177
9.3.2.3 COMMENTS ON THE ELECTROPHORETIC PURIFICATION AND ELUTION OF RNA
SPECIES 178
9.3.3 ISOLATION OF SINGLE RNA SPECIES WITH PARTIALLY KNOWN
SEQUENCE 178
9.3.3.1 MATERIALS FOR HYBRID SELECTION OF SINGLE RNA SPECIES 178
9.4 RADIOACTIVE LABELING OF RNA TERMINI 180
9.4.1 MATERIALS FOR 5'-END LABELING OF RNAS 180
9.4.2 3'-LABELING OF RNAS 181
9.4.2.1 MATERIALS FOR 3'-END LABELING OF RNAS 182
9.5 SEQUENCING OF END-LABELED RNA 183
9.5.1 SEQUENCING BY BASE-SPECIFIC ENZYMATIC HYDROLYSIS OF END-LABELED
RNA 184
9.5.1.1 MATERIALS REQUIRED FOR ENZYMATIC SEQUENCING 185
9.5.1.2 INTERPRETATION AND TROUBLESHOOTING 186
9.5.2 SEQUENCING BY BASE-SPECIFIC CHEMICAL MODIFICATION AND
CLEAVAGE 187
9.5.2.1 MATERIALS REQUIRED FOR CHEMICAL SEQUENCING 188
X I CONTENTS
9.5.2.2 INTERPRETATION AND TROUBLESHOOTING 189
9.6 DETERMINATION OF TERMINAL RNA SEQUENCES BY TWO-DIMENSIONAL
MOBILITY SHIFT 190
9.6.1 MATERIALS REQUIRED FOR MOBILITY SHIFT ANALYSIS 190
9.7 DETERMINATION OF MODIFIED NUCLEOTIDES BY POSTLABELING
METHODS 194
9.7.1 ANALYSIS OF TOTAL NUCLEOTIDE CONTENT 195
9.7.1.1 MATERIALS REQUIRED FOR RNA NUCLEOTIDE ANALYSIS 195
9.7.1.2 INTERPRETATION AND TROUBLESHOOTING 197
9.7.2 DETERMINATION OF POSITION AND IDENTITY OF MODIFIED
NUCLEOTIDES 198
9.7.2.1 INTERPRETATION AND TROUBLESHOOTING 199
9.8 CONCLUSIONS AND OUTLOOK 201
ACKNOWLEDGMENTS 202
REFERENCES 202
10 PROBING RNA STRUCTURE IN VITRO WITH ENZYMES AND CHEMICALS 205
ANNE-CATHERINE HEIFER, C6DRIC
ROMILLY, CLDMENT CHEVALIER,
EJTHIMIA LIOLIOU, STEFANO MARZI, AND PASCALE ROMBY
10.1 INTRODUCTION 205
10.2 ENZYMATIC AND CHEMICAL PROBES 207
10.2.1 ENZYMES 207
10.2.2 BASE-SPECIFIC CHEMICAL PROBES 210
10.2.3 BACKBONE-SPECIFIC CHEMICAL PROBES 211
10.3 IN VIVO DMS MODIFICATION 222
10.3.1 GENERALITIES 222
10.3.2 IN VIVO PROBING 222
10.4 COMMENTARY 223
10.4.1 CRITICAL PARAMETERS 223
10.4.1.1 RNA PREPARATION 223
10.4.1.2 HOMOGENEOUS RNA CONFORMATION 224
10.4.1.3 CHEMICAL AND ENZYMATIC PROBING 224
10.4.1.4 IN VIVO DMS MAPPING 225
10.5 TROUBLESHOOTING 225
ACKNOWLEDGMENTS 227
REFERENCES 227
11 PROBING RNA SOLUTION STRUCTURE BY PHOTOCROSSLINKING: INCORPORATION
OF PHOTOREACTIVE CROUPS AT RNA TERMINI AND DETERMINATION OF
CROSSLINKED SITES BY PRIMER EXTENSION 231
MICHAEL E. HARRIS
11.1 INTRODUCTION 231
11.1.1 APPLICATIONS OF RNA MODIFICATIONS 231
11.1.2 TECHNIQUES FOR THE INCORPORATION OF MODIFIED NUCLEOTIDES 232
11.2 DESCRIPTION 233
CONTENTS | XI
11.2.1 5'-END MODIFICATION BY TRANSCRIPTION PRIMING 233
11.2.2 CHEMICAL PHOSPHORYLATION OF NUCLEOSIDES TO GENERATE
5'-MONOPHOSPHATE OR 5'-MONOPHOSPHOROTHIOATE DERIVATIVES 234
11.2.3 ATTACHMENT OF AN ARYL AZIDE PHOTOCROSSLINKING AGENT TO A
5'-TERMINAL
PHOSPHOROTHIOATE 236
11.2.4 3'-ADDITION OF AN ARYL AZIDE PHOTOCROSSLINKING AGENT 238
11.3 TROUBLESHOOTING 240
11.4 PROBING RNA STRUCTURE BY PHOTOAFFINITY CROSSLINKING WITH
4-THIOURIDINE AND 6-THIOGUANOSINE 240
11.4.1 INTRODUCTION 240
11.4.2 DESCRIPTION 243
11.4.2.1 GENERAL CONSIDERATIONS: REACTION CONDITIONS AND CONCENTRATIONS
OF INTERACTING SPECIES 243
11.4.2.2 APPLICATION EXAMPLE - RNASE P RNA AND S
6
G-MODIFIED PRECURSOR
TRNA 244
11.4.2.3 GENERATION AND ISOLATION OF CROSSLINKED RNAS 246
11.4.2.4 PRIMER EXTENSION MAPPING OF CROSSLINKED NUCLEOTIDES 247
11.4.3 TROUBLESHOOTING 249
REFERENCES 250
12 TERBIUM(LLL) FOOTPRINTING AS A PROBE OF RNA STRUCTURE AND METAL
BINDING SITES 255
DINARI A. HARRIS, GABRIELLE C. TODD,
AND NILS G. WALTER
12.1 INTRODUCTION 255
12.2 APPLICATION EXAMPLE 261
12.3 TROUBLESHOOTING 265
12.4 FRONTIERS IN FOOTPRINTING DATA ANALYSIS 265
REFERENCES 266
13 PB
2+
-LNDUCED CLEAVAGE OF RNA 269
LEIF A. KIRSEBOM AND JERZY CIESIOLKA
13.1 INTRODUCTION 269
13.2 PB
2+
-INDUCED CLEAVAGE TO PROBE METAL ION BINDING SITES, RNA
STRUCTURE, AND RNA-LIGAND INTERACTIONS 271
13.2.1 PROBING HIGH-AFFINITY METAL ION BINDING SITES 271
13.2.2 PB
2+
-INDUCED CLEAVAGE AND RNA STRUCTURE 273
13.2.3 PB
2+
-INDUCED CLEAVAGE TO STUDY RNA-LIGAND
INTERACTIONS 274
13.2.4 PB
2+
-INDUCED CLEAVAGE OF RNA IN VIVO 275
13.3 TROUBLESHOOTING 279
13.3.1 NO PB
2+
-INDUCED CLEAVAGE DETECTED 279
13.3.2 COMPLETE DEGRADATION OF THE RNA 280
13.3.3 IN VIVO 280
ACKNOWLEDGMENTS 280
REFERENCES 281
XII CONTENTS
14 IDENTIFICATION AND CHARACTERIZATION OF METAL ION COORDINATION
INTERACTIONS WITH RNA BY QUANTITATIVE ANALYSIS OF THIOPHILIC METAL
ION RESCUE OF SITE-SPECIFIC PHOSPHOROTHIOATE MODIFICATIONS 285
MICHAEL E. HARRIS
14.1 INTRODUCTION 285
14.1.1 THIOPHILIC METAL ION RESCUE OF RNA PHOSPHOROTHIOATE
MODIFICATIONS 286
14.2 PURIFICATION OF PHOSPHOROTHIOATE STEREOISOMERS BY RP-HPLC 290
14.3 TECHNIQUES FOR INCORPORATION OF PHOSPHOROTHIOATES INTO RNA 291
14.4 KINETIC ANALYSIS OF THIOPHILIC METAL ION RESCUE 293
14.5 DATA ANALYSIS BY FITTING TO SIMPLE EQUILIBRIUM MODELS 295
REFERENCES 297
15 PROBING RNA STRUCTURE AND LIGAND BINDING SITES ON RNA BY FENTON
CLEAVAGE 301
CORINA G. HEIDRICH AND CHRISTIAN BERENS
15.1 INTRODUCTION 301
15.2 COMMENTS AND TROUBLESHOOTING 312
REFERENCES 314
16 MEASURING THE STOICHIOMETRY OF MAGNESIUM IONS BOUND TO RNA 319
ANDREW J. ANDREWS AND CAROL A. FIERKE
16.1 INTRODUCTION 319
16.2 SEPARATION OF FREE MG
2+
FROM RNA-BOUND MG
2+
320
16.3 FORCED DIALYSIS IS THE PREFERRED METHOD FOR SEPARATING BOUND
AND FREE MG
2+
321
16.4 ALTERNATIVE METHODS FOR SEPARATING FREE AND BOUND MG
2+
IONS 323
16.5 DETERMINING THE CONCENTRATION OF FREE MG
2+
IN THE
FLOW-THROUGH 324
16.6 HOW TO DETERMINE THE CONCENTRATION OF MG
2+
BOUND TO THE RNA
AND THE NUMBER OF BINDING SITES ON THE RNA 324
16.7 CONCLUSION 327
16.8 TROUBLESHOOTING 327
REFERENCES 327
17 NUCLEOTIDE ANALOG INTERFERENCE MAPPING AND SUPPRESSION
(NAIM/NAIS): A COMBINATORIAL APPROACH TO STUDY RNA STRUCTURE,
FOLDING, AND INTERACTION
WITH PROTEINS 329
OLGA FEDOROVA, MARC
BOUDVILLAIN, AND CHRISTINA WALDSICH
17.1 INTRODUCTION 329
17.1.1 NAIM: A COMBINATORIAL APPROACH FOR RNA STRUCTURE-FUNCTION
ANALYSIS 329
17.1.1.1 DESCRIPTION OF THE METHOD 330
17.1.2 NAIS: A CHEMOGENETIC TOOL FOR IDENTIFYING RNA TERTIARY CONTACTS
AND INTERACTION INTERFACES 332
CONTENTS XIII
17.1.2.1 GENERAL CONCEPTS 332
17.1.2.2 APPLICATIONS: ELUCIDATING TERTIARY CONTACTS IN GROUP I AND
GROUP II
RIBOZYMES 332
17.2 EXPERIMENTAL PROTOCOLS FOR NAIM 333
17.2.1 NUCLEOSIDE ANALOG THIOTRIPHOSPHATES 333
17.2.2 PREPARATION OF TRANSCRIPTS CONTAINING PHOSPHOROTHIOATE
ANALOGS 335
17.2.2.1 TIPS AND TROUBLESHOOTING 336
17.2.3 RADIOACTIVE LABELING OF THE RNA POOL 337
17.2.4 THE SELECTION STEP OF NAIM: THREE APPLICATIONS TO STUDIES OF RNA
FUNCTION 339
17.2.4.1 GROUP II INTRON RIBOZYME ACTIVITY: SELECTION THROUGH
TRANSESTERIFICATION 339
17.2.4.2 GROUP II RIBOZYME FOLDING: SELECTION THROUGH MG
2+
-INDUCED
COMPACTION OF RNA 344
17.2.4.3 RNA-PROTEIN INTERACTIONS: A ONE-POT REACTION FOR STUDYING
RHO-INDEPENDENT TRANSCRIPTION TERMINATION 347
17.2.4.4 RNA-PROTEIN INTERACTIONS: ELUCIDATION OF THE RHO HELICASE
ACTIVATION
MECHANISM VIA UNWINDING ACTIVITY 351
17.2.5 IODINE CLEAVAGE OF RNA POOLS 354
17.2.5.1 EXPERIMENTAL PROCEDURE 355
17.2.5.2 TIPS AND TROUBLESHOOTING 355
17.2.6 ANALYSIS AND INTERPRETATION OF NAIM RESULTS 355
17.2.6.1 QUANTIFICATION OF INTERFERENCE EFFECTS 355
17.3 EXPERIMENTAL PROTOCOLS FOR NAIS 358
17.3.1 DESIGN AND CONSTRUCTION OF RNA MUTANTS 358
17.3.1.1 GENERAL CONSIDERATIONS 358
17.3.1.2 PREPARATION OF RNA MOLECULES CONTAINING SINGLE-ATOM
SUBSTITUTIONS 359
17.3.2 FUNCTIONAL ANALYSIS OF MUTANTS FOR NAIS EXPERIMENTS 362
17.3.3 THE SELECTION STEP FOR NAIS 362
17.3.4 DATA ANALYSIS AND PRESENTATION 363
ACKNOWLEDGMENTS 364
REFERENCES 364
18 NUCLEOTIDE ANALOG INTERFERENCE MAPPING (NAIM): APPLICATION TO THE
RNASE P SYSTEM 369
SIMONA CUZIC-FELTENS AND ROLAND K. HARTMANN
18.1 INTRODUCTION 369
18.1.1 NUCLEOTIDE ANALOG INTERFERENCE MAPPING (NAIM) - THE
APPROACH 369
18.1.2 CRITICAL ASPECTS OF THE METHOD 371
18.1.2.1 ANALOG INCORPORATION 371
18.1.2.2 FUNCTIONAL ASSAYS 372
18.1.2.3 FACTORS INFLUENCING THE OUTCOME OF NAIM STUDIES 372
XIV
| CONTENTS
18.1.3 INTERPRETATION OF RESULTS 373
18.2 NAIM ANALYSIS OF CIS-CLEAVING RNASE P RNA-TRNA CONJUGATES 375
18.2.1 BIOCHEMICAL AND KINETIC CHARACTERIZATION OF A CIS-CLEAVING E.
COLI
RNASE P RNA-TRNA CONJUGATE 375
18.2.2 APPLICATION EXAMPLE 378
18.2.3 DATA EVALUATION 386
18.3 TROUBLESHOOTING 387
18.3.1 RNA TRANSCRIPTION REACTION DID NOT WORK 387
18.3.2 RNA DEGRADATION 389
18.3.3 INEFFICIENT RNA ELUTION FROM DENATURING PAA GELS 389
18.3.4 RNA IS DEGRADED AFTER ELUTION 389
18.3.5 INEFFICIENT 3'- OR 5'-END-LABELING 389
18.3.6 IODINE-INDUCED HYDROLYSIS FAILED OR WAS INEFFICIENT 391
18.3.7 UNSATISFACTORY GEL PERFORMANCE AFTER IODINE CLEAVAGE (BAND
SMEARING, CURVED BANDS, IRREGULAR SHAPE OF BANDS, UNEQUAL BAND
MIGRATION IN DIFFERENT LANES, AND INSUFFICIENT BAND SEPARATION) 392
REFERENCES 393
19 IDENTIFICATION OF DIVALENT METAL ION BINDING SITES IN
RNA/DNA-METABOLIZING ENZYMES BY FE(LL)-MEDIATED HYDROXY
I RADICAL
CLEAVAGE 397
YAN-GUO REN, NIKLAS HENRIKSSON, AND ANDERS VIRTANEN
19.1 INTRODUCTION 397
19.2 PROBING DIVALENT METAL ION BINDING SITES 398
19.2.1 FE(II)-MEDIATED HYDROXYL RADICAL CLEAVAGE 398
19.2.2 HOW TO MAP DIVALENT METAL ION BINDING SITES 399
19.2.3 HOW TO USE AMINOGLYCOSIDES AS FUNCTIONAL AND STRUCTURAL
PROBES 401
19.3 NOTES AND TROUBLESHOOTING 403
REFERENCES 404
20 RNA STRUCTURE AND FOLDING ANALYZED USING SMALL-ANGLE X-RAY
SCATTERING 407
NATHAN J. BAIRD,
JEREMEY WEST, AND TOBIN R. SOSNICK
20.1 INTRODUCTION 407
20.2 DESCRIPTION OF METHOD 410
20.2.1 GENERAL REQUIREMENTS 410
20.2.2 SAXS APPLICATION EXAMPLE 411
20.2.3 GENERAL INFORMATION 412
20.2.4 QUESTION 1: THE GLOBAL CONFORMATION OF THE S-DOMAIN FOLDING
INTERMEDIATE 412
20.2.5 QUESTION 2: THE STABLE, EXTENDED CONFORMATION OF THE S-DOMAIN
FOLDING INTERMEDIATE 414
20.2.6 QUESTION 3: THE UTILITY OF LOW-RESOLUTION REAL-SPACE
RECONSTRUCTIONS
IN RNA MODELING 416
CONTENTS I XV
20.3 TROUBLESHOOTING 421
20.3.1 PROBLEM 1: RADIATION DAMAGE AND AGGREGATION 421
20.3.2 PROBLEM 2: HIGH SCATTERING BACKGROUND 422
20.3.3 PROBLEM 3: SCATTERING RESULTS CANNOT BE FIT TO SIMPLE MODELS 422
20.4 CONCLUSIONS - OUTLOOK 422
ACKNOWLEDGMENTS 423
ABBREVIATIONS 423
REFERENCES 423
21 TEMPERATURE-GRADIENT GEL ELECTROPHORESIS OF RNA 427
DETLEV
RIESNER AND GERHARD
STEGER
21.1 INTRODUCTION 427
21.2 METHOD 428
21.2.1 PRINCIPLE 428
21.2.2 INSTRUMENTS 429
21.2.3 HANDLING 429
21.3 OPTIMIZATION OF EXPERIMENTAL CONDITIONS 430
21.3.1 PORE SIZE OF THE GEL MATRIX 430
21.3.2 ELECTRIC FIELD 430
21.3.3 IONIC STRENGTH AND UREA 431
21.4 TGGE - GENERAL INTERPRETATION RULES 431
21.5 EXAMPLES OF TGGE APPLICATIONS 433
21.5.1 EXAMPLE 1: ANALYSIS OF DIFFERENT RNA MOLECULES IN A SINGLE
TGGE 434
21.5.2 EXAMPLE 2: ANALYSIS OF STRUCTURE TRANSITIONS IN A SINGLE RNA -
DETECTION OF SPECIFIC STRUCTURES BY OLIGONUCLEOTIDE
HYBRIDIZATION
435
21.5.3 EXAMPLE 3: ANALYSIS OF MUTANTS 438
21.5.4 EXAMPLE 4: DETECTION OF PROTEIN-RNA COMPLEXES BY TGGE 439
21.5.5 OUTLOOK 442
REFERENCES 443
22 UV MELTING STUDIES WITH RNA 445
PHILIPPE DUMAS, ERIC
ENNIFAR, FRANCOIS DISDIER, AND PHILIPPE WALTER
22.1 INTRODUCTION 445
22.2 A SIMPLIFIED ACCOUNT OF THE PHYSICAL BASIS OF UV ABSORPTION 445
22.3 DEFINITIONS AND NOMENCLATURE 446
22.4 WELL-KNOWN AND LESS WELL-KNOWN CHARACTERISTICS OF UV ABSORPTION
BY NUCLEIC ACIDS BASES 447
22.5 THE BASIS OF UV MELTING EXPERIMENTS FOR THERMODYNAMIC
STUDIES 449
22.5.1 THE ONLY VALID DEFINITION OF A MELTING TEMPERATURE 450
22.5.2 REMINDERS 450
22.5.3 UNIMOLECULAR TRANSITIONS 451
22.5.4 BIMOLECULAR TRANSITIONS 452
XVII CONTENTS
22.5.4.1 ENTROPIC CONSIDERATIONS 452
22.5.4.2 BASIC AND LESS BASIC EQUATIONS ABOUT MELTING CURVES INVOLVING
BIMOLECULAR TRANSITIONS 454
22.5.4.3 HIGHER ORDER TRANSITIONS 455
22.5.4.4 INFLUENCE OF THE TEMPERATURE DEPENDENCE OF THE ABSORBANCE
PARAMETERS 455
22.5.4.5 THE DIFFERENT WAYS OF OBTAINING T
M
, AH, AND AS 455
22.6 THE TWO-STATE APPROXIMATION AND ITS LIMITATIONS 459
22.7 EQUILIBRIUM AND NON-EQUILIBRIUM 459
22.8 A COMMON PITFALL WITH SELF-COMPLEMENTARY SEQUENCES 460
22.9 EXTRACTING THERMODYNAMIC INFORMATION FROM MELTING CURVES OF
LARGE RNAS 461
22.10 PARAMETERS INFLUENCING THE MELTING TEMPERATURE 462
22.11 PRACTICAL PROBLEMS 463
22.11.1 EVAPORATION DURING HEATING: AN IMPORTANT IMPROVEMENT 463
22.11.2 SLOPING BASELINE 464
22.12 A NEAT EXPERIMENTAL SOLUTION TO THE SLOPING BASELINE 468
22.12.1 PH VARIATION AND BUFFERS 468
22.12.2 RNA DEGRADATION 470
22.12.3 HEATING RATE AND DATA SAMPLING 471
22.12.4 EXPERIMENTAL DATA PROCESSING 472
22.12.5 SOFTWARES 473
ACKNOWLEDGMENT 473
APPENDIX A: DIFFERENCE BETWEEN T
M
AND T
MAX
AND DMC
NORMALIZATION 473
APPENDIX B: EXPERIMENTAL SETUP AGAINST EVAPORATION 475
APPENDIX C: THE SUBTLETIES WITH PARTIAL DERIVATIVES FOR ACP
DETERMINATION 475
APPENDIX D: BUFFER PK
A
VARIATION WITH THE TEMPERATURE 476
REFERENCES 476
RNA CRYSTALLIZATION 481
JIRO KONDO, CLAUDE SAUTER, AND BENOIT MASQUIDA
INTRODUCTION 481
RNA PURIFICATION 482
HPLC PURIFICATION 482
GEL ELECTROPHORESIS 483
RNA RECOVERY 484
ELUTION OF THE RNA FROM THE GEL 484
CONCENTRATING AND DESALTING 484
RNA CRYSTALLIZATION 485
RENATURING THE RNA 485
SEARCH FOR CRYSTALLIZATION CONDITIONS 485
EVALUATION OF CRYSTALLIZATION ASSAYS 488
THE OPTIMIZATION PROCESS 489
23
23.1
23.2
23.2.1
23.2.2
23.2.3
23.2.3.1
23.2.3.2
23.3
23.3.1
23.3.2
23.3.3
23.3.4
CONTENTS | XVII
23.3.5 DESIGNING RNA CONSTRUCTS WITH IMPROVED CRYSTALLIZATION
CAPABILITIES 491
23.3.6 CRYSTALLIZING COMPLEXES WITH ORGANIC LIGANDS: THE EXAMPLE OF
AMINOGLYCOSIDES 493
23.4 CONCLUSIONS 494
REFERENCES 495
24 STUDYING RNA USING SINGLE MOLECULE FLUORESCENCE RESONANCE ENERGY
TRANSFER 499
FELIX SPENKUCH, OLWEN DOMINGO, GERALD HINZE, THOMAS BASCHI,
AND MARK HELM
24.1 INTRODUCTION 499
24.1.1 THE ADVANTAGES OF SINGLE MOLECULE FLUORESCENCE RESONANCE ENERGY
TRANSFER 499
24.1.2 CHAPTER SCOPE 500
24.1.3 TYPICAL TOPICS OF RNA DYNAMICS ADDRESSED BY SINGLE MOLECULE
FRET 500
24.2 THEORY OF FLUORESCENCE RESONANCE ENERGY TRANSFER 502
24.3 EXPERIMENTAL DESIGN 503
24.3.1 CONSIDERATIONS FOR CONSTRUCT DESIGN 503
24.4 SMFRET EXPERIMENTS USING IMMOBILIZED MOLECULES 505
24.4.1 INSTRUMENTAL SETUP 505
24.4.2 MEANS OF SIGNAL CORRECTION AND DATA ANALYSIS 505
24.4.3 THE CHOICE OF DYE PAIRS FOR FRET 507
24.4.4 BUFFER HANDLING IN SINGLE MOLECULE EXPERIMENTS 508
24.4.5 STRATEGIES FOR DYE LABELING OF RNA CONSTRUCTS 508
24.4.6 POSTSYNTHETIC LABELING OF ALKYNE-CONTAINING RNA
OLIGONUCLEOTIDES 509
24.4.7 TUNING DYE ENDURANCE: ANTIFADING AGENTS 510
24.5 TROUBLESHOOTING 520
24.5.1 RNASE CONTAMINATION 520
24.5.2 REMOVAL OF UNBOUND FLUOROPHORES 521
24.5.3 DRYING OF SAMPLES 521
24.5.4 DONOR-ONLY POPULATIONS 521
24.5.5 TOO DENSE OR TOO SPARSE SURFACE COVERAGE 521
REFERENCES 522
25 ATOMIC FORCE MICROSCOPY IMAGING AND FORCE SPECTROSCOPY OF
RNA 527
MALTE BUSSIEK, ANTONIE SCHONE, AND WOLFGANG NELLEN
25.1 INTRODUCTION 527
25.2 AFM IMAGING OF RNA STRUCTURES 528
25.2.1 GENERAL PRECONDITIONS: MODE OF OPERATION, DATA ANALYSIS, AND
RESOLUTION 528
25.2.2 SURFACE PREPARATION CONDITIONS 531
XVIII CONTENTS
25.2.3 IMAGING IN LIQUID 535
25.2.4 EXPERIMENTAL EXAMPLE OF SALT-DEPENDENT RNA FOLDING USING A
DESIGNED RNA CONSTRUCT 535
25.3 EXAMPLE PROTOCOL: RNA PREPARATION FOR AFM IMAGING IN AIR USING
PL-COATED MICA 537
25.4 TROUBLESHOOTING 538
25.5 FORCE SPECTROSCOPY AFM 540
25.6 OUTLOOK 544
ACKNOWLEDGMENTS 544
REFERENCES 544
PART III RNA GENOMICS & BIOINFORMATICS, GLOBAL APPROACHES 547
26 SECONDARY STRUCTURE PREDICTION 549
GERHARD STEGER
26.1 INTRODUCTION 549
26.2 THERMODYNAMICS 550
26.3 FORMAL BACKGROUND 552
26.4 MF OLD AND UNAFOLD 555
26.4.1 INPUT TO THE MF OLD SERVER 556
26.4.1.1 SEQUENCE NAME 556
26.4.1.2 SEQUENCE 556
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 MF OLD 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 RNAF
OLD 565
26.5.1 INPUT TO THE RNAF OLD 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 RNAF OLD 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
CONTENTS | XIX
27 RNA SECONDARY STRUCTURE ANALYSIS USING ABSTRACT SHAPES 579
ROBERT GIEGERICH AND
BJOM VOFI
27.1 INTRODUCTION TO ABSTRACT SHAPE ANALYSIS 579
27.1.1 LOOKING DEEPER INTO THE RNA FOLDING SPACE 579
27.1.2 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) 581
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
27.3 PROTOCOL 2: PROBABILISTIC SHAPE ANALYSIS 585
27.3.1 USEFUL PARAMETERS 587
27.4 PROTOCOL 3: COMPARATIVE SHAPE ANALYSIS FROM ALIGNED
SEQUENCES 587
27.4.1 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 615
XX | CONTENTS
28.8 PROGRAM VERSIONS AND DOWNLOAD SITES 616
ACKNOWLEDGMENTS 616
REFERENCES 616
29 HOMOLOGY SEARCH FOR SMALL STRUCTURED NON-CODING RNAS 619
MANJA MARZ, STEFANIE WEHNER, AND PETER F. STADLER
29.1 INTRODUCTION 619
29.2 MATERIALS 619
29.2.1 SEQUENCE DATA 619
29.2.2 WEB SERVICES 620
29.2.3 WEB SERVICE-INDEPENDENT SOFTWARE 621
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
30 PREDICT RNA 2D AND 3D STRUCTURE OVER THE INTERNET USING
MC-TOOLS 633
STEPHEN LEONG KOAN,
JONATHAN ROY, MARC PARISIEN, AND FRANCOIS
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 AND ASSEMBLE2 673
REFERENCES 685
CONTENTS XXI
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
X-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
32.3.4
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
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 701
32.4.1 EVALUATING THE QUALITY OF THE TRAJECTORIES 701
32.4.1.1 CONSISTENCY CHECKS 702
XXII
I CONTENTS
32.4.1.2 COMPARISON WITH EXPERIMENTAL DATA 702
32.4.1.3 VISUALIZATION 702
32.4.1.4 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
32.4.4.1 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 J ORG VOGEL
33.1 INTRODUCTION 719
33.2 EXPRESSION-BASED DISCOVERY OF SRNAS 720
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 SRNA 728
33.4.1 SRNA EXPRESSION PROFILE 729
33.4.2 SRNA DELETION 729
33.4.3 SRNA 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 SRNA 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
BJORN VOJI AND WOLFGANG R. HESS
34.1 INTRODUCTION 787
CONTENTS | XXIII
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
35 EXPERIMENTAL RNOMICS, A GLOBAL APPROACH TO IDENTIFY NON-CODING
RNAS IN MODEL ORGANISMS, AND RNPOMICS TO ANALYZE THE NON-CODING
RNPTRANSCRIPTOME 801
MATHIEU REDERSTORFF AND
ALEXANDER HTITTENHOFER
35.1 INTRODUCTION 801
35.2 COMPUTATIONAL ANALYSIS OF NCRNA SEQUENCES 811
35.3 NOTES 812
35.4 COMPUTATIONAL ANALYSIS OF NCRNA SEQUENCES
816
35.5 NOTES 816
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
XXIV
| CONTENTS
36.3 PROTOCOLS: USEFUL ALGORITHMS, FORMATS, AND TOOLS 830
REFERENCES 830
37 CHARACTERIZATION AND PREDICTION OF MIRNA TARGETS 833
JEAN HAUSSER AND MIHAELA ZAVOLAN
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
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 CONSISTENDY 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 X 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 X 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
CONTENTS I XXV
38 BARCODED CDNA LIBRARIES FOR MIRNA PROFILING BY NEXT-GENERATION
SEQUENCING 861
MARKUS HAJHER, 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-RIBONUDEOSIDE-ENHANCED CROSSLINKING AND
IMMUNOPRECIPITATION)
877
JESSICA
I. HOELL, MARKUS HAJHER, MARKUS LANDTHALER, MANUEL ASCANO,
THALIA A. FARAZI, GREG WARDLE, JEFF NUSBAUM, PAVOL CEKAN,
MOHSEN KHORSHID,
LUKAS BURGER,
MIHAELA ZAVOLAN, AND THOMAS TUSCHL
39.1 INTRODUCTION 877
39.2 TROUBLESHOOTING 897
ACKNOWLEDGMENTS 897
REFERENCES 897
40 GLOBAL ANALYSIS OF PROTEIN-RNA INTERACTIONS WITH SINGLE-NUDEOTIDE
RESOLUTION USING ICLIP 899
JULIAN KONIG, NICHOLAS J. MC GLINCY, AND JERNEJ 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 OLIGONUCLEOTIDE DESIGN 903
40.2.4 TROUBLESHOOTING 904
ACKNOWLEDGMENTS 917
REFERENCES 917
PART IV RNA FUNCTION, RN P ANALYSIS,
SELEX, RNAI 919
41 USE OF RNA AFFINITY MATRICES FOR THE ISOLATION OF RNA BINDING
PROTEINS 921
MARKUS ENGLERT,
BETTINA SPATH, STEFFEN SCHIFFER, SYLVIA ROSCH,
HILDBURG BEIER, AND ANITA MARCH/ELDER
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
XXVI
|
CONTENTS
41.3
NOTES 932
REFERENCES 932
42 BIOTIN-BASED AFFINITY
PURIFICATION OF RNA-PROTEIN COMPLEXES 935
MARCO PREUJINER, SILKE
SCHREINER, INNA GRISHINA, ZSOFIA 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 OFTOTAL CELL LYSATE 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
42.3.1.4 ELUTION OF AFFINITY-SELECTED RNPS FOR FUNCTIONAL STUDIES BY A
DISPLACEMENT OLIGONUCLEOTIDE 945
42.3.2 AFFINITY PURIFICATION OF SPECIFIC RNA BINDING PROTEINS BY
BIOTINYLATED
RNAS 948
42.3.3 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
42.4.4 OPTIMIZATION OF THE EXPERIMENTAL CONDITIONS IN THE CASE OF HIGH
BACKGROUND 953
REFERENCES 953
43 AFFINITY PURIFICATION OF SPLICEOSOMAL AND SMALL NUCLEAR
RIBONUDEOPROTEIN COMPLEXES 957
JULIA DANNENBERG, PATRIZIA FABRIZIO, CINDY L. WILL,
AND REINHARD LIIHRMANN
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 SPLICEOSOMAL COMPLEXES 963
ACKNOWLEDGMENTS 971
REFERENCES 972
CONTENTS XXVII
44 STUDY OF RNA-PROTEIN INTERACTIONS AND RNA STRUCTURE IN
RIBONUDEOPROTEIN PARTICLES (RNPS) 975
VIRGINIE MARCHAND, ANNIE MOUGIN, AGNSS MIREAU, 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
44.2.4 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
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 993
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
44.3.1.1 RNA PURIFICATION AND RENATURATION 1005
44.3.1.2 EMSA 1005
44.3.1.3 TOBRAMYCIN-SEPHAROSE 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
XXVIII
| CONTENTS
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 ANKO AND KARLA M. NEUGEBAUER
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
45.2.2.2 RT-PCR ANALYSIS OF IMMUNOPURIFIED RNA 1024
45.2.2.3 NEXT-GENERATION SEQUENCING OF IMMUNOPURIFIED 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 1027
ACKNOWLEDGMENTS 1027
REFERENCES 1027
46 PROTEIN-RNA CROSSLINKING IN NATIVE RIBONUDEOPROTEIN PARTICLES 1029
OLEXANDR DYBKOV, HENNING URLAUB, AND REINHARD LIIHRMANN
46.1 INTRODUCTION 1029
46.2 OVERALL STRATEGY 1030
46.3 UV CROSSLINKING 1031
46.4 IDENTIFICATION OF UV-INDUCED PROTEIN-RNA CROSSLINKING SITES BY
PRIMER EXTENSION ANALYSIS 1033
46.5 IDENTIFICATION OF CROSSLINKED PROTEINS 1037
46.6 TROUBLESHOOTING 1040
ACKNOWLEDGMENTS 1050
REFERENCES 1050
47 SEDIMENTATION ANALYSIS OF RIBONUDEOPROTEIN COMPLEXES 1055
TANJA ROSEL, JAN MEDENBACH, ANDREY DAMIANOV, SILKE SCHREINER, AND
ALBRECHT BINDEREIF
47.1 INTRODUCTION 1055
CONTENTS XXIX
47.2 GLYCEROL GRADIENT CENTRIFUGATION 1056
47.3 FRACTIONATION OF RIBONUCLEOPROTEINS (RNPS) BY CESIUM CHLORIDE
DENSITY GRADIENT CENTRIFUGATION 1061
REFERENCES 1065
48 IDENTIFICATION AND CHARACTERIZATION OF RNA BINDING PROTEINS THROUGH
THREE-HYBRID ANALYSIS 1067
FELICIA SCOTT
AND DAVID R. ENGELKE
48.1 INTRODUCTION 1067
48.2 BASIC STRATEGY OF THE METHOD 1068
48.3 DETAILED COMPONENTS 1070
48.3.1 YEAST REPORTER STRAIN 1070
48.3.2 PLASMIDS 1070
48.3.3 HYBRID RNA 1071
48.3.3.1 TECHNICAL CONSIDERATIONS FOR THE HYBRID RNA 1071
48.3.4 ACTIVATION DOMAIN FP2 1073
48.3.4.1 TECHNICAL CONSIDERATIONS FOR THE ACTIVATION DOMAIN OF FP2 1074
48.3.5 POSITIVE CONTROLS 1075
48.4 TROUBLESHOOTING 1079
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 1100
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
XXX I CONTENTS
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 DISULFIDE-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 1150
51.4.1 REVERSE TRANSCRIPTION 1151
51.4.2 POLYMERASE CHAIN REACTION 1152
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 ISOCRATIC COMPETITIVE AFFINITY CHROMATOGRAPHY 1161
REFERENCES 1162
52 SELEX STRATEGIES TO IDENTIFY ANTISENSE AND PROTEIN TARGET SITES IN
RNA
OR KNRNP COMPLEXES 1165
MARTIN LUTZELBERGER, MARTIN R. JAKOBSEN, AND JORGEN 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 1172
CONTENTS | XXXI
52.6.3 INEFFICIENT MME I DIGESTION 1172
52.6.4 THE AMPLIFICATION OF THE UNSELECTED LIBRARY IS INEFFICIENT 1173
52.6.5 THE LIBRARY APPEARS TO BE NON-RANDOM IN THE UNSELECTED POOL 1173
52.6.6 THE SELECTED RNAS DO NOT BIND TO NATIVE PROTEIN 1173
REFERENCES 1182
53 GENOMIC SELEX 1185
JENNIFER I. BOOTS, KATARZYNA MATYLLA-KULINSKA, MARCH ZYWICKI,
BOB ZIMMERMANN, AND RENSE SCHROEDER
53.1 INTRODUCTION 1185
53.2 DESCRIPTION OF THE METHODS 1186
53.2.1 LIBRARY CONSTRUCTION 1186
53.2.2 CHOICE OF BAIT 1188
53.2.3 SELEX PROCEDURE 1188
53.2.3.1 TRANSCRIPTION OF GENOMIC LIBRARY INTO RNA LIBRARY 1190
53.2.3.2 COUNTER SELECTION 1190
53.2.3.3 POSITIVE SELECTION 1190
53.2.3.4 RECOVERY AND AMPLIFICATION OF SELECTED SEQUENCES 1191
53.2.3.5 NEUTRAL SELEX 1192.
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 BIOCHEMICAL 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 1202
REFERENCES 1202
54 IN 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 1210
54.2.2 DESIGN OF THE MINIGENE 1212
XXXII
| CONTENTS
54.2.3 RT-PCR AMPLIFICATION 1213
54.2.4 MONITORING FOR ENRICHMENT OF EXON SEQUENCES THAT FUNCTION AS
SPLICING ENHANCERS 1213
54.2.5 TROUBLESHOOTING 1214
ACKNOWLEDGMENTS 1218
REFERENCES 1219
55 CENE SILENCING METHODS USING VECTOR-ENCODED SIRNAS OR
MIRNAS 1221
YING POI LIU AND BEN BERKHOUT
55.1 INTRODUCTION 1221
55.2 BACKGROUND INFORMATION 1221
55.3 CONSTRUCTION OF SHRNA VECTORS 1223
55.4 CONSTRUCTION OF MIRNA VECTORS 1228
55.5 CONSTRUCTION OF EXTENDED SHRNAS AND LHRNAS 1229
55.6 PRODUCTION OF LENTIVIRAL VECTORS ENCODING ANTI-HIV-1 SHRNAS OR
E-SHRNAS 1230
55.6.1 TROUBLESHOOTING 1234
REFERENCES 1235
56 USING CHEMICAL MODIFICATION TO ENHANCE SIRNA PERFORMANCE 1243
JESPER B. BRAMSEN, ARNOLD
GRIINWELLER, ROLAND K. HARTMANN,
AND JORGEN 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 1252
56.6 ENHANCING SIRNA NUCLEASE RESISTANCE 1253
56.6.1 SIRNA STABILITY AND RIBONUDEASES 1253
56.6.2 STRATEGIES FOR SIRNA STABILIZATION 1254
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 1257
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
CONTENTS XXXIII
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 1261
56.12 A GUIDE FOR IN VIVO STUDIES 1261
REFERENCES 1265
APPENDIX: UV SPECTROSCOPY FOR THE QUANTITATION OF RNA 1279
INDEX 1283 |
| any_adam_object | 1 |
| author2 | Hartmann, Roland K. 1956- |
| author2_role | edt |
| author2_variant | r k h rk rkh |
| author_GND | (DE-588)12993643X |
| author_facet | Hartmann, Roland K. 1956- |
| building | Verbundindex |
| bvnumber | BV042755974 |
| classification_rvk | WD 5355 |
| ctrlnum | (OCoLC)884883590 (DE-599)DNB1067782060 |
| 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 |
| edition | 2., completely rev. and enl. ed. |
| format | Book |
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| genre | (DE-588)4127380-1 Praktikum gnd-content |
| genre_facet | Praktikum |
| id | DE-604.BV042755974 |
| illustrated | Illustrated |
| indexdate | 2024-08-03T02:35:56Z |
| institution | BVB |
| isbn | 3527327762 9783527327768 9783527647064 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-028186616 |
| oclc_num | 884883590 |
| open_access_boolean | |
| owner | DE-29T DE-703 DE-188 DE-355 DE-BY-UBR |
| owner_facet | DE-29T DE-703 DE-188 DE-355 DE-BY-UBR |
| physical | LIV, 1314 S. Ill., graph. Darst. 24 cm |
| publishDate | 2014 |
| publishDateSearch | 2014 |
| publishDateSort | 2014 |
| publisher | Wiley-VCH |
| record_format | marc |
| spelling | Handbook of RNA biochemistry ed. by Roland K. Hartmann ... 2., completely rev. and enl. ed. Weinheim Wiley-VCH 2014 LIV, 1314 S. Ill., graph. Darst. 24 cm txt rdacontent n rdamedia nc rdacarrier Literaturangaben RNS (DE-588)4076759-0 gnd rswk-swf Biochemie Molekularbiologie Ribonukleinsäure (DE-588)4127380-1 Praktikum gnd-content RNS (DE-588)4076759-0 s DE-604 Hartmann, Roland K. 1956- (DE-588)12993643X edt 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 B:DE-101 application/pdf http://d-nb.info/1067782060/04 Inhaltsverzeichnis X:MVB text/html http://deposit.dnb.de/cgi-bin/dokserv?id=5165445&prov=M&dok_var=1&dok_ext=htm Inhaltstext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028186616&sequence=000001&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)4127380-1 |
| 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 K. Hartmann ... |
| title_fullStr | Handbook of RNA biochemistry ed. by Roland K. Hartmann ... |
| title_full_unstemmed | Handbook of RNA biochemistry ed. by Roland K. Hartmann ... |
| title_short | Handbook of RNA biochemistry |
| title_sort | handbook of rna biochemistry |
| topic | RNS (DE-588)4076759-0 gnd |
| topic_facet | RNS Praktikum |
| url | http://d-nb.info/1067782060/04 http://deposit.dnb.de/cgi-bin/dokserv?id=5165445&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=028186616&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT hartmannrolandk handbookofrnabiochemistry |
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