Edited by Roland K. Hartmann,

Albrecht Bindereif, Astrid Schon,

and Eric Westhof

Handbook of RNA Biochemistry

Related Titles

Meister, G.

RNA BiologyAn Introduction

2011

ISBN: 978-3-527-32278-7

Gjerde, D. T., Hoang, L., Hornby, D.

RNA Purification and AnalysisSample Preparation, Extraction,Chromatography

2009

ISBN: 978-3-527-32116-2

Gu, J., Bourne, P. E. (eds.)

Structural Bioinformatics

2009

ISBN: 978-0-470-18105-8

Edited by Roland K. Hartmann, Albrecht Bindereif,Astrid Schon, and Eric Westhof

Handbook of RNA Biochemistry

Second, Completely Revised and Enlarged Edition

The Editors

Prof. Dr. Roland K. HartmannPhilipps-Universitat MarburgInstitut fur Pharma. ChemieMarbacher Weg 635037 MarburgGermany

Prof. Dr. Albrecht BindereifJustus-Liebig-UniversitatInstitut fur BiochemieHeinrich-Buff-Ring 5835392 GießenGermany

Dr. Astrid SchonUniversitat LeipzigMolecular Cell TherapyDeutscher Platz 504103 LeipzigGermany

Prof. Dr. Eric WesthofCNRS - UPR 9002, Inst. deBiol. Mol. et Cellulaire15 rue Rene Descartes06708 StrasbourgFrance

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Printed in SingaporePrinted on acid-free paper

V

Contents

Preface XXXVList of Contributors XXXVII

Part I RNA Synthesis and Detection 1

1 Enzymatic RNA Synthesis Using Bacteriophage T7 RNA Polymerase 3Markus Goßringer, Dominik Helmecke, Karen Kohler, Astrid Schon,Leif A. Kirsebom, Albrecht Bindereif, and Roland K. Hartmann

1.1 Introduction 31.2 Description of Method – T7 Transcription In vitro 41.2.1 Templates 41.2.1.1 Strategy (i): Insertion into a Plasmid 41.2.1.2 Strategy (ii): Direct Use of Templates Generated by PCR 51.2.1.3 Strategy (iii): Annealing of a T7 Promoter DNA Oligonucleotide

to a Single-Stranded Template 51.2.2 Special Demands on the RNA Product 51.2.2.1 Homogeneous 5′ and 3′ Ends, Small RNAs, Functional Groups

at the 5′ End 51.2.2.2 Modified Substrates 61.3 Transcription Protocols 81.3.1 Transcription with Unmodified Nucleotides 91.3.2 Transcription with 2′-Fluoro-Modified Nucleotides 161.3.3 T7 Transcripts with 5′-Cap Structures 171.3.4 Purification 181.4 Troubleshooting 201.4.1 Low or No Product Yield 201.5 Rapid Preparation of T7 RNA Polymerase 211.5.1 Required Material 211.5.1.1 Medium 211.5.1.2 Buffers and Solutions 211.5.1.3 Electrophoresis and Chromatography 221.5.2 Procedure 221.5.2.1 Cell Growth, Induction, and Test for Expression of T7 RNAP 22

VI Contents

1.5.2.2 Purification of T7 RNAP 231.5.3 Notes and Troubleshooting 24

References 25

2 Production of RNAs with Homogeneous 5′- and 3′-Ends 29Mario Morl and Roland K. Hartmann

2.1 Introduction 292.2 Description of Approach 302.2.1 Cis-Cleaving Autocatalytic Ribozyme Cassettes 302.2.1.1 The 5′-Cassette 302.2.1.2 The 3′-Cassette 302.2.1.3 Purification of Released RNA Product and Conversion of End

Groups 312.2.2 Trans-Cleaving Ribozymes for the Generation of Homogeneous

3′ Ends 332.2.3 Further Strategies toward Homogeneous Ends 352.3 Critical Experimental Steps, Changeable Parameters,

Troubleshooting 362.3.1 Construction of Cis-Cleaving 5′- and 3′-Cassettes 362.4 PCR Protocols 372.5 Potential Problems 42

References 42

3 RNA Ligation 45Janne J. Turunen, Liudmila V. Pavlova, Martin Hengesbach, Mark Helm,Sabine Muller, Roland K. Hartmann, and Mikko J. Frilander

3.1 General Introduction 453.1.1 T4 Polynucleotide Ligases 463.1.2 Reaction Mechanism 463.1.3 Advantages of T4 DNA Ligase for RNA Ligation 493.1.4 Chapter Structure 493.2 RNA Ligation Using T4 DNA Ligase (T4 Dnl) 503.2.1 Overview of the RNA Ligation Method Using the T4 DNA Ligase

(T4 Dnl) 513.2.2 Large-Scale Transcription and Purification of RNAs 533.2.3 Generating Homogeneous Acceptor 3′-Ends for Ligation 533.2.4 Site-Directed Cleavage with RNase H 543.2.5 Dephosphorylation and Phosphorylation of RNAs 563.2.6 RNA Ligation 573.2.7 Troubleshooting 583.3 Simultaneous Splint Ligation of Five RNA Fragments to Generate

RNAs for FRET Experiments 663.3.1 Introduction 663.3.2 Construct Design 683.3.3 Troubleshooting 70

Contents VII

3.3.3.1 Low Overall Ligation Efficiency 703.3.3.2 Undesired Ligation By-products 703.3.3.3 RNA Degradation 703.4 T4 RNA Ligase(s) 703.4.1 Introduction 703.4.2 Mechanism and Substrate Specificity 713.4.2.1 Early Studies 713.4.2.2 Substrate Specificity and Reaction Conditions 723.4.3 Applications of T4 RNA Ligase 733.4.3.1 End-Labeling 733.4.3.2 Circularization 753.4.3.3 Intermolecular Ligation of Polynucleotides 753.4.4 T4 RNA Ligation of Large RNA Molecules 763.4.5 Application Examples and Protocols 793.4.5.1 Production of Full-Length tRNAs 793.4.6 Troubleshooting 84

References 84

4 Northern Blot Detection of Small RNAs 89Benedikt M. Beckmann, Arnold Grunweller, and Roland K. Hartmann

4.1 Introduction 894.1.1 Isolation of RNA 894.1.1.1 Kits 904.1.1.2 Do it Yourself 904.1.1.3 Quality Control 904.1.2 Native versus Denaturing Gels 904.1.3 Transfer of RNA and Fixation to Membranes 914.1.4 Hybridization with a Complementary Probe 924.1.4.1 Design of DNA/LNA Mixmer Probes 924.1.5 Detection of DIG-Labeled Probes 954.1.6 Troubleshooting 954.1.7 Application Example 964.1.8 Limitations of the Method 964.2 Northern Hybridization Protocols 98

References 102

5 Rapid, Non-Denaturing, Large-Scale Purification of In Vitro TranscribedRNA Using Weak Anion-Exchange Chromatography 105Laura E. Easton, Yoko Shibata, and Peter J. Lukavsky

5.1 Introduction 1055.2 Materials 1065.2.1 Cloning and Plasmid Purification 1065.2.2 In Vitro Transcription 1065.2.3 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 115Acknowledgments 115References 116

6 3′-Terminal Attachment of Fluorescent Dyes and Biotin 117Dagmar K. Willkomm and Roland K. Hartmann

6.1 Introduction 1176.2 Description of Method 1186.3 History of the Method 1186.4 Troubleshooting 1246.4.1 Problems Caused Before the Labeling Reaction 1246.4.1.1 Quality of the RNA 3′ Ends 1246.4.1.2 Purity of the RNA to Be Labeled 1246.4.2 Problems with the Labeling Reaction Itself 1246.4.2.1 pH of Reagents 1246.4.2.2 Stability of Reagents 1246.4.3 Postlabeling Problems 1256.4.3.1 Removal of Labeling Reagents 1256.4.3.2 Loss of RNA Material during Downstream Purification 1256.4.3.3 Stability of Labeled RNA 125

Acknowledgment 125References 125

7 Chemical RNA Synthesis, Purification, and Analysis 129Brian S. Sproat

7.1 Introduction 1297.2 Description 1327.2.1 The Solid-Phase Synthesis of RNA 1327.2.2 Deprotection 1367.2.3 Purification 1387.2.3.1 Anion-Exchange HPLC Purification 1397.2.3.2 Reversed-Phase HPLC Purification of Trityl-On RNA 1407.2.3.3 Detritylation of Trityl-On RNA 1427.2.3.4 Desalting by HPLC 1427.2.4 Analysis of the Purified RNA 1437.3 Troubleshooting 144

References 147

8 Modified RNAs as Tools in RNA Biochemistry 151Thomas E. Edwards and Snorri Th. Sigurdsson

8.1 Introduction 1518.1.1 Modification Strategy: the Phosphoramidite Method 1528.1.2 Modification Strategy: Postsynthetic Labeling 154

Contents IX

8.2 Description of Methods 1568.2.1 Postsynthetic Modification: the 2′-Amino Approach 1568.2.2 Reaction of 2′-Amino Groups with Succinimidyl Esters 1588.2.3 Reaction of 2′-Amino Groups with Aromatic Isothiocyanates 1588.2.4 Reaction of 2′-Amino Groups with Aliphatic Isocyanates 1598.3 Experimental Protocols 1598.3.1 Synthesis of Aromatic Isothiocyanates and Aliphatic

Isocyanates 1608.3.2 Postsynthetic Labeling of 2′-Amino-Modified RNA 1618.3.3 Postsynthetic Labeling of 4-Thiouridine-Modified RNA 1648.3.4 Verification of Label Incorporation 1648.3.5 Potential Problems and Troubleshooting 165

References 166

Part II Structure Determination 173

9 Direct Determination of RNA Sequence and Modification byRadiolabeling Methods 175Olaf Gimple and Astrid Schon

9.1 Introduction 1759.2 General Methods 1759.3 Isolation of Pure RNA Species from Biological Material 1769.3.1 Preparation of Size-Fractionated RNA 1769.3.2 Isolation of a Single Unknown RNA Species Following a Functional

Assay 1769.3.2.1 Solutions for Electrophoresis, Staining, and Elution of RNAs from

Gels 1769.3.2.2 Two-Dimensional Electrophoresis of RNA 1779.3.2.3 Comments on the Electrophoretic Purification and Elution of RNA

Species 1789.3.3 Isolation of Single RNA Species with Partially Known

Sequence 1789.3.3.1 Materials for Hybrid Selection of Single RNA Species 1789.4 Radioactive Labeling of RNA Termini 1809.4.1 Materials for 5′-End Labeling of RNAs 1809.4.2 3′-Labeling of RNAs 1819.4.2.1 Materials for 3′-End Labeling of RNAs 1829.5 Sequencing of End-Labeled RNA 1839.5.1 Sequencing by Base-Specific Enzymatic Hydrolysis of End-Labeled

RNA 1849.5.1.1 Materials Required for Enzymatic Sequencing 1859.5.1.2 Interpretation and Troubleshooting 1869.5.2 Sequencing by Base-Specific Chemical Modification and

Cleavage 1879.5.2.1 Materials Required for Chemical Sequencing 188

X Contents

9.5.2.2 Interpretation and Troubleshooting 1899.6 Determination of Terminal RNA Sequences by Two-dimensional

Mobility Shift 1909.6.1 Materials Required for Mobility Shift Analysis 1909.7 Determination of Modified Nucleotides by Postlabeling

Methods 1949.7.1 Analysis of Total Nucleotide Content 1959.7.1.1 Materials Required for RNA Nucleotide Analysis 1959.7.1.2 Interpretation and Troubleshooting 1979.7.2 Determination of Position and Identity of Modified

Nucleotides 1989.7.2.1 Interpretation and Troubleshooting 1999.8 Conclusions and Outlook 201

Acknowledgments 202References 202

10 Probing RNA Structure In Vitro with Enzymes and Chemicals 205Anne-Catherine Helfer, Cedric Romilly, Clement Chevalier,Efthimia Lioliou, Stefano Marzi, and Pascale Romby

10.1 Introduction 20510.2 Enzymatic and Chemical Probes 20710.2.1 Enzymes 20710.2.2 Base-Specific Chemical Probes 21010.2.3 Backbone-Specific Chemical Probes 21110.3 In Vivo DMS Modification 22210.3.1 Generalities 22210.3.2 In Vivo Probing 22210.4 Commentary 22310.4.1 Critical Parameters 22310.4.1.1 RNA Preparation 22310.4.1.2 Homogeneous RNA Conformation 22410.4.1.3 Chemical and Enzymatic Probing 22410.4.1.4 In Vivo DMS Mapping 22510.5 Troubleshooting 225

Acknowledgments 227References 227

11 Probing RNA Solution Structure by Photocrosslinking: Incorporationof Photoreactive Groups at RNA Termini and Determination ofCrosslinked Sites by Primer Extension 231Michael E. Harris

11.1 Introduction 23111.1.1 Applications of RNA Modifications 23111.1.2 Techniques for the Incorporation of Modified Nucleotides 23211.2 Description 233

Contents XI

11.2.1 5′-End Modification by Transcription Priming 23311.2.2 Chemical Phosphorylation of Nucleosides to Generate

5′-Monophosphate or 5′-Monophosphorothioate Derivatives 23411.2.3 Attachment of an Aryl Azide Photocrosslinking Agent to a 5′-Terminal

Phosphorothioate 23611.2.4 3′-Addition of an Aryl Azide Photocrosslinking Agent 23811.3 Troubleshooting 24011.4 Probing RNA Structure by Photoaffinity Crosslinking with

4-Thiouridine and 6-Thioguanosine 24011.4.1 Introduction 24011.4.2 Description 24311.4.2.1 General Considerations: Reaction Conditions and Concentrations

of Interacting Species 24311.4.2.2 Application Example – RNase P RNA and s6G-Modified Precursor

tRNA 24411.4.2.3 Generation and Isolation of Crosslinked RNAs 24611.4.2.4 Primer Extension Mapping of crosslinked Nucleotides 24711.4.3 Troubleshooting 249

References 250

12 Terbium(III) Footprinting as a Probe of RNA Structure and MetalBinding Sites 255Dinari A. Harris, Gabrielle C. Todd, and Nils G. Walter

12.1 Introduction 25512.2 Application Example 26112.3 Troubleshooting 26512.4 Frontiers in Footprinting Data Analysis 265

References 266

13 Pb2+-Induced Cleavage of RNA 269Leif A. Kirsebom and Jerzy Ciesiolka

13.1 Introduction 26913.2 Pb2+-Induced Cleavage to Probe Metal Ion Binding Sites, RNA

Structure, and RNA–Ligand Interactions 27113.2.1 Probing High-Affinity Metal Ion Binding Sites 27113.2.2 Pb2+-Induced Cleavage and RNA Structure 27313.2.3 Pb2+-Induced Cleavage to Study RNA–Ligand

Interactions 27413.2.4 Pb2+-Induced Cleavage of RNA In Vivo 27513.3 Troubleshooting 27913.3.1 No Pb2+-Induced Cleavage Detected 27913.3.2 Complete Degradation of the RNA 28013.3.3 In Vivo 280

Acknowledgments 280References 281

XII Contents

14 Identification and Characterization of Metal Ion CoordinationInteractions with RNA by Quantitative Analysis of Thiophilic MetalIon Rescue of Site-Specific Phosphorothioate Modifications 285Michael E. Harris

14.1 Introduction 28514.1.1 Thiophilic Metal Ion Rescue of RNA Phosphorothioate

Modifications 28614.2 Purification of Phosphorothioate Stereoisomers by RP-HPLC 29014.3 Techniques for Incorporation of Phosphorothioates into RNA 29114.4 Kinetic Analysis of Thiophilic Metal Ion Rescue 29314.5 Data Analysis by Fitting to Simple Equilibrium Models 295

References 297

15 Probing RNA Structure and Ligand Binding Sites on RNA by FentonCleavage 301Corina G. Heidrich and Christian Berens

15.1 Introduction 30115.2 Comments and Troubleshooting 312

References 314

16 Measuring the Stoichiometry of Magnesium Ions Bound to RNA 319Andrew J. Andrews and Carol A. Fierke

16.1 Introduction 31916.2 Separation of Free Mg2+ from RNA-bound Mg2+ 32016.3 Forced Dialysis Is the Preferred Method for Separating Bound

and Free Mg2+ 32116.4 Alternative Methods for Separating Free and Bound Mg2+ Ions 32316.5 Determining the Concentration of Free Mg2+ in the

Flow-Through 32416.6 How to Determine the Concentration of Mg2+ Bound to the RNA

and the Number of Binding Sites on the RNA 32416.7 Conclusion 32716.8 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 329Olga Fedorova, Marc Boudvillain, and Christina Waldsich

17.1 Introduction 32917.1.1 NAIM: a Combinatorial Approach for RNA Structure–Function

Analysis 32917.1.1.1 Description of the Method 33017.1.2 NAIS: a Chemogenetic Tool for Identifying RNA Tertiary Contacts

and Interaction Interfaces 332

Contents XIII

17.1.2.1 General Concepts 33217.1.2.2 Applications: Elucidating Tertiary Contacts in Group I and Group II

Ribozymes 33217.2 Experimental Protocols for NAIM 33317.2.1 Nucleoside Analog Thiotriphosphates 33317.2.2 Preparation of Transcripts Containing Phosphorothioate

Analogs 33517.2.2.1 Tips and Troubleshooting 33617.2.3 Radioactive Labeling of the RNA Pool 33717.2.4 The Selection Step of NAIM: Three Applications to Studies of RNA

Function 33917.2.4.1 Group II Intron Ribozyme Activity: Selection through

Transesterification 33917.2.4.2 Group II Ribozyme Folding: Selection through Mg2+-Induced

Compaction of RNA 34417.2.4.3 RNA–Protein Interactions: a One-Pot Reaction for Studying

Rho-Independent Transcription Termination 34717.2.4.4 RNA–Protein Interactions: Elucidation of the Rho Helicase Activation

Mechanism via Unwinding Activity 35117.2.5 Iodine Cleavage of RNA Pools 35417.2.5.1 Experimental Procedure 35517.2.5.2 Tips and Troubleshooting 35517.2.6 Analysis and Interpretation of NAIM Results 35517.2.6.1 Quantification of Interference Effects 35517.3 Experimental Protocols for NAIS 35817.3.1 Design and Construction of RNA Mutants 35817.3.1.1 General Considerations 35817.3.1.2 Preparation of RNA Molecules Containing Single-Atom

Substitutions 35917.3.2 Functional Analysis of Mutants for NAIS Experiments 36217.3.3 The Selection Step for NAIS 36217.3.4 Data Analysis and Presentation 363

Acknowledgments 364References 364

18 Nucleotide Analog Interference Mapping (NAIM): Application to theRNase P System 369Simona Cuzic-Feltens and Roland K. Hartmann

18.1 Introduction 36918.1.1 Nucleotide Analog Interference Mapping (NAIM) – the

Approach 36918.1.2 Critical Aspects of the Method 37118.1.2.1 Analog Incorporation 37118.1.2.2 Functional Assays 37218.1.2.3 Factors Influencing the Outcome of NAIM Studies 372

XIV Contents

18.1.3 Interpretation of Results 37318.2 NAIM Analysis of cis-Cleaving RNase P RNA-tRNA Conjugates 37518.2.1 Biochemical and kinetic characterization of a cis-Cleaving E. coli

RNase P RNA-tRNA Conjugate 37518.2.2 Application Example 37818.2.3 Data Evaluation 38618.3 Troubleshooting 38718.3.1 RNA Transcription Reaction Did Not Work 38718.3.2 RNA Degradation 38918.3.3 Inefficient RNA Elution from Denaturing PAA Gels 38918.3.4 RNA Is Degraded after Elution 38918.3.5 Inefficient 3′- or 5′-End-Labeling 38918.3.6 Iodine-Induced Hydrolysis Failed or Was Inefficient 39118.3.7 Unsatisfactory Gel Performance after Iodine Cleavage (Band

Smearing, Curved Bands, Irregular Shape of Bands, Unequal BandMigration in Different Lanes, and Insufficient Band Separation) 392References 393

19 Identification of Divalent Metal Ion Binding Sites inRNA/DNA-Metabolizing Enzymes by Fe(II)-Mediated Hydroxyl RadicalCleavage 397Yan-Guo Ren, Niklas Henriksson, and Anders Virtanen

19.1 Introduction 39719.2 Probing Divalent Metal Ion Binding Sites 39819.2.1 Fe(II)-Mediated Hydroxyl Radical Cleavage 39819.2.2 How to Map Divalent Metal Ion Binding Sites 39919.2.3 How to Use Aminoglycosides as Functional and Structural

Probes 40119.3 Notes and Troubleshooting 403

References 404

20 RNA Structure and Folding Analyzed Using Small-Angle X-RayScattering 407Nathan J. Baird, Jeremey West, and Tobin R. Sosnick

20.1 Introduction 40720.2 Description of Method 41020.2.1 General Requirements 41020.2.2 SAXS Application Example 41120.2.3 General Information 41220.2.4 Question 1: The Global Conformation of the S-Domain Folding

Intermediate 41220.2.5 Question 2: The Stable, Extended Conformation of the S-Domain

Folding Intermediate 41420.2.6 Question 3: The Utility of Low-Resolution Real-Space Reconstructions

in RNA Modeling 416

Contents XV

20.3 Troubleshooting 42120.3.1 Problem 1: Radiation Damage and Aggregation 42120.3.2 Problem 2: High Scattering Background 42220.3.3 Problem 3: Scattering Results Cannot Be Fit to Simple Models 42220.4 Conclusions – Outlook 422

Acknowledgments 423Abbreviations 423References 423

21 Temperature-Gradient Gel Electrophoresis of RNA 427Detlev Riesner and Gerhard Steger

21.1 Introduction 42721.2 Method 42821.2.1 Principle 42821.2.2 Instruments 42921.2.3 Handling 42921.3 Optimization of Experimental Conditions 43021.3.1 Pore Size of the Gel Matrix 43021.3.2 Electric Field 43021.3.3 Ionic Strength and Urea 43121.4 TGGE – General Interpretation Rules 43121.5 Examples of TGGE Applications 43321.5.1 Example 1: Analysis of Different RNA Molecules in a Single

TGGE 43421.5.2 Example 2: Analysis of Structure Transitions in a Single RNA –

Detection of Specific Structures by OligonucleotideHybridization 435

21.5.3 Example 3: Analysis of Mutants 43821.5.4 Example 4: Detection of Protein–RNA Complexes by TGGE 43921.5.5 Outlook 442

References 443

22 UV Melting Studies with RNA 445Philippe Dumas, Eric Ennifar, Francois Disdier, and Philippe Walter

22.1 Introduction 44522.2 A Simplified Account of the Physical Basis of UV Absorption 44522.3 Definitions and Nomenclature 44622.4 Well-Known and Less Well-Known Characteristics of UV Absorption

by Nucleic Acids Bases 44722.5 The Basis of UV Melting Experiments for Thermodynamic

Studies 44922.5.1 The Only Valid Definition of a Melting Temperature 45022.5.2 Reminders 45022.5.3 Unimolecular Transitions 45122.5.4 Bimolecular Transitions 452

XVI Contents

22.5.4.1 Entropic Considerations 45222.5.4.2 Basic and Less Basic Equations about Melting Curves Involving

Bimolecular Transitions 45422.5.4.3 Higher Order Transitions 45522.5.4.4 Influence of the Temperature Dependence of the Absorbance

Parameters 45522.5.4.5 The Different Ways of Obtaining Tm, �H, and �S 45522.6 The Two-State Approximation and Its Limitations 45922.7 Equilibrium and Non-equilibrium 45922.8 A Common Pitfall with Self-Complementary Sequences 46022.9 Extracting Thermodynamic Information from Melting Curves of

Large RNAs 46122.10 Parameters Influencing the Melting Temperature 46222.11 Practical Problems 46322.11.1 Evaporation during Heating: an Important Improvement 46322.11.2 Sloping Baseline 46422.12 A Neat Experimental Solution to the Sloping Baseline 46822.12.1 pH Variation and Buffers 46822.12.2 RNA Degradation 47022.12.3 Heating Rate and Data Sampling 47122.12.4 Experimental Data Processing 47222.12.5 Softwares 473

Acknowledgment 473Appendix A: Difference between Tm and Tmax and DMCNormalization 473Appendix B: Experimental Setup against Evaporation 475Appendix C: The Subtleties with Partial Derivatives for �CP

Determination 475Appendix D: Buffer pKa Variation with the Temperature 476References 476

23 RNA Crystallization 481Jiro Kondo, Claude Sauter, and Benoıt Masquida

23.1 Introduction 48123.2 RNA Purification 48223.2.1 HPLC Purification 48223.2.2 Gel Electrophoresis 48323.2.3 RNA Recovery 48423.2.3.1 Elution of the RNA from the Gel 48423.2.3.2 Concentrating and Desalting 48423.3 RNA Crystallization 48523.3.1 Renaturing the RNA 48523.3.2 Search for Crystallization Conditions 48523.3.3 Evaluation of Crystallization Assays 48823.3.4 The Optimization Process 489

Contents XVII

23.3.5 Designing RNA Constructs with Improved CrystallizationCapabilities 491

23.3.6 Crystallizing Complexes with Organic Ligands: the Example ofAminoglycosides 493

23.4 Conclusions 494References 495

24 Studying RNA Using Single Molecule Fluorescence Resonance EnergyTransfer 499Felix Spenkuch, Olwen Domingo, Gerald Hinze, Thomas Basche,and Mark Helm

24.1 Introduction 49924.1.1 The Advantages of Single Molecule Fluorescence Resonance Energy

Transfer 49924.1.2 Chapter Scope 50024.1.3 Typical Topics of RNA Dynamics Addressed by Single Molecule

FRET 50024.2 Theory of Fluorescence Resonance Energy Transfer 50224.3 Experimental Design 50324.3.1 Considerations for Construct Design 50324.4 smFRET Experiments Using Immobilized Molecules 50524.4.1 Instrumental Setup 50524.4.2 Means of Signal Correction and Data Analysis 50524.4.3 The Choice of Dye Pairs for FRET 50724.4.4 Buffer Handling in Single Molecule Experiments 50824.4.5 Strategies for Dye Labeling of RNA Constructs 50824.4.6 Postsynthetic Labeling of Alkyne-Containing RNA

Oligonucleotides 50924.4.7 Tuning Dye Endurance: Antifading Agents 51024.5 Troubleshooting 52024.5.1 RNase Contamination 52024.5.2 Removal of Unbound Fluorophores 52124.5.3 Drying of Samples 52124.5.4 Donor-Only Populations 52124.5.5 Too Dense or Too Sparse Surface Coverage 521

References 522

25 Atomic Force Microscopy Imaging and Force Spectroscopy ofRNA 527Malte Bussiek, Antonie Schone, and Wolfgang Nellen

25.1 Introduction 52725.2 AFM Imaging of RNA Structures 52825.2.1 General Preconditions: Mode of Operation, Data Analysis, and

Resolution 52825.2.2 Surface Preparation Conditions 531

XVIII Contents

25.2.3 Imaging in Liquid 53525.2.4 Experimental Example of Salt-Dependent RNA Folding Using a

Designed RNA Construct 53525.3 Example Protocol: RNA Preparation for AFM Imaging in Air Using

PL-Coated Mica 53725.4 Troubleshooting 53825.5 Force Spectroscopy AFM 54025.6 Outlook 544

Acknowledgments 544References 544

Part III RNA Genomics & Bioinformatics, Global Approaches 547

26 Secondary Structure Prediction 549Gerhard Steger

26.1 Introduction 54926.2 Thermodynamics 55026.3 Formal Background 55226.4 mfold and UNAFold 55526.4.1 Input to the mfold Server 55626.4.1.1 Sequence Name 55626.4.1.2 Sequence 55626.4.1.3 Constraints 55626.4.1.4 Further Parameters 55826.4.1.5 Immediate versus Batch Jobs 56126.4.2 Output from the mfold Server 56126.4.2.1 Energy Dot Plot 56126.4.2.2 Extra Files 56326.4.2.3 Download All Foldings 56326.4.2.4 View ss-Count Information 56426.4.2.5 View Individual Structures 56426.4.2.6 Dot Plot Folding Comparisons 56526.5 RNAfold 56526.5.1 Input to the RNAfold Server 56626.5.1.1 Sequence and Constraints 56626.5.1.2 Further Parameters 56726.5.1.3 Immediate versus Batch Jobs 56826.5.2 Output from the RNAfold Server 57026.5.2.1 Text Output of Secondary Structure 57026.5.2.2 Probability Dot Plot 57026.5.2.3 Graphical Output of Secondary Structure 57026.5.2.4 Mountain Plot 57126.6 Troubleshooting 571

Acknowledgment 573References 573

Contents XIX

27 RNA Secondary Structure Analysis Using Abstract Shapes 579Robert Giegerich and Bjorn Voß

27.1 Introduction to Abstract Shape Analysis 57927.1.1 Looking Deeper into the RNA Folding Space 57927.1.2 Overview of Functions of Abstract Shape Analysis 58027.1.3 Definition of Shape Abstraction 58027.1.3.1 Shapes 58027.1.3.2 Shape Abstraction Function 58127.1.3.3 Shape Representative Structures (shreps) 58127.1.3.4 Levels of Abstraction 58127.1.3.5 Shape Probabilities 58227.1.3.6 Consensus Shape 58227.1.4 General Caveats when Working with Abstract

Shapes 58227.1.5 Applications of Abstract Shape Analysis 58327.2 Protocol 1: Computing Shape Representative

Structures 58427.2.1 Useful Parameters for RNAshapes 58527.3 Protocol 2: Probabilistic Shape Analysis 58527.3.1 Useful Parameters 58727.4 Protocol 3: Comparative Shape Analysis from Aligned

Sequences 58727.4.1 Useful Parameters for RNAlishapes 58827.5 Protocol 4: Comparative Shape Analysis from Unaligned

Sequences 58827.5.1 Useful Parameters for RNAshapes 59227.6 RNAshapes Parameter Overview 59227.7 RNAlishapes Parameter Overview 593

References 594

28 Screening Genome Sequences for known RNA Genes or Motifs 595Daniel Gautheret

28.1 Introduction 59528.2 Choosing the Right Search Program 59628.3 Overview of the RNA Search Procedure 59728.4 Assessing Search Specificity 59828.5 A Test Case: Looking for Homologs of a Bacterial sRNA 60028.5.1 Building a First Training Set with BLASTN 60028.5.2 Alignment and Structure Prediction 60228.5.3 Searching with HMMER 60428.5.4 Searching with RNAMOTIF 60628.5.5 Searching with ERPIN 60928.5.6 Searching with INFERNAL 61428.6 Conclusion 61528.7 Supplemental Data 615

XX Contents

28.8 Program Versions and Download Sites 616Acknowledgments 616References 616

29 Homology Search for Small Structured Non-coding RNAs 619Manja Marz, Stefanie Wehner, and Peter F. Stadler

29.1 Introduction 61929.2 Materials 61929.2.1 Sequence Data 61929.2.2 Web Services 62029.2.3 Web Service-Independent Software 62129.3 Protocol: mascRNAs 62129.3.1 The Seed 62229.3.2 Low-Hanging Fruits: Initial BLAST Search 62329.3.3 Initial Secondary Structure Model 62429.3.4 Drilling Deep – Structure-Based Searches 62529.4 Concluding Remarks 629

Acknowledgments 630References 630

30 Predict RNA 2D and 3D Structure over the Internet UsingMC-Tools 633Stephen Leong Koan, Jonathan Roy, Marc Parisien, and Francois Major

30.1 Introduction 63330.2 Materials 63430.2.1 Equipment 63430.2.2 Data 63430.3 MC-Tools 63530.3.1 MC-Fold 63530.3.2 MC-Cons 63630.3.3 MC-Sym 63630.4 Troubleshooting 663

Acknowledgments 663References 663

31 S2S-Assemble2: a Semi-Automatic Bioinformatics Framework to Studyand Model RNA 3D Architectures 667Fabrice Jossinet and Eric Westhof

31.1 Introduction 66731.2 S2S: an Interactive RNA Alignment Viewer and Editor 66831.3 Assemble2: an Interactive RNA 3D Modeler 67131.4 The Semi-Automatic Architecture of S2S and Assemble2 67231.5 Installation of S2S and Assemble2 673

References 685

Contents XXI

32 Molecular Dynamics Simulations of RNA Systems 687Pascal Auffinger

32.1 Introduction 68732.2 MD Methods 68932.3 Simulation Setups 68932.3.1 Selecting an Appropriate Starting Structure 68932.3.1.1 Model-Built Structures 68932.3.1.2 X-Ray and Neutron Diffraction Structures 68932.3.1.3 Cryo-Electron Microscopy (Cryo-EM) Structures 69032.3.1.4 NMR Structures 69032.3.2 Checking the Starting Structure 69032.3.2.1 Conformational Checks 69032.3.2.2 Rare Non-covalent Interactions 69132.3.2.3 Protonation Issues 69232.3.2.4 Solvent 69232.3.3 Adding Hydrogen Atoms 69332.3.4 Choosing the Environment (Crystal, Liquid) and Ion Types 69332.3.5 Setting the Box Size and Placing the Ions and Water 69332.3.5.1 Box Size 69332.3.5.2 Monovalent Ions 69332.3.5.3 Divalent Ions 69432.3.5.4 Minimal Salt Conditions 69432.3.5.5 Water Molecules 69432.3.5.6 Building Initial Solute and Solvent Configurations 69432.3.6 Choosing the Program and Force Field 69532.3.6.1 Programs 69532.3.6.2 Force Fields 69532.3.6.3 Parameterization of Modified Nucleotides, Ligands, and Ions 69632.3.6.4 Clustering Artifacts and Ion Parameters 69632.3.6.5 Water Models 69632.3.7 Treatment of Electrostatic Interactions 69732.3.8 Other Simulation Parameters 69732.3.8.1 Thermodynamic Ensemble 69732.3.8.2 Temperature and Pressure 69832.3.8.3 Shake, Time Steps, and Update of the Non-bonded Pair List 69832.3.8.4 The ‘‘Flying Ice Cube Problem’’ 69832.3.9 Equilibration 69932.3.10 Sampling 69932.3.10.1 How Long Should a Simulation Be? 69932.3.10.2 When to Stop a Simulation 70032.3.10.3 Multiple Molecular Dynamics (MMD) Simulations 70132.3.10.4 Simulations of Large Systems 70132.4 Analysis 70132.4.1 Evaluating the Quality of the Trajectories 70132.4.1.1 Consistency Checks 702

XXII Contents

32.4.1.2 Comparison with Experimental Data 70232.4.1.3 Visualization 70232.4.1.4 Validation through Statistical Survey of Structural Databases 70332.4.2 Convergence Issues 70332.4.3 Conformational Parameters 70332.4.4 Data Analysis 70432.4.4.1 Clustering 70432.4.4.2 Analysis Packages 70432.4.4.3 Solvent Analysis 70432.5 Perspectives 704

Acknowledgments 705References 705

33 Identification and Characterization of Small Non-coding RNAsin Bacteria 719Dimitri Podkaminski, Marie Bouvier, and Jorg Vogel

33.1 Introduction 71933.2 Expression-Based Discovery of sRNAs 72033.2.1 Microarray 72033.2.2 High-Throughput Sequencing and RNA-Seq 72133.2.3 Hfq Coimmunoprecipitation 72433.3 Expression-Independent Searches 72633.3.1 Biocomputational Approaches 72633.3.2 Genomic SELEX 72833.4 Deciphering the Biological Role of an sRNA 72833.4.1 sRNA Expression Profile 72933.4.2 sRNA Deletion 72933.4.3 sRNA Overexpression 73133.4.4 sRNA Pulse Expression Combined with Transcriptome

Analysis 73333.4.5 sRNA Libraries 73433.4.6 Finding sRNA-Associated Proteins 73533.4.7 Biocomputational Approaches to Find Targets 73633.5 Experimental Target Validation 73733.5.1 Reporter Gene Fusions and sRNA Chimera 73833.5.2 In vitro RNA–RNA Footprinting 73933.5.3 In vitro Characterization of sRNA Function 74133.6 Conclusions 742

Acknowledgments 776References 776

34 The Identification of Bacterial Non-coding RNAs throughComplementary Approaches 787Bjorn Voß and Wolfgang R. Hess

34.1 Introduction 787

Contents XXIII

34.2 Computational Prediction 78734.2.1 Workflow 78834.2.2 Results and Interpretation 78934.2.3 Alternative Approaches 79034.2.4 Troubleshooting 79134.2.4.1 Choice of Genomes 79134.2.4.2 Short mRNAs and Dual-Function RNAs 79434.3 Experimental Approaches for High-Throughput RNomics

in Bacteria 79434.3.1 Microarray Analysis 79434.3.1.1 Considerations for the Design of Tiling Microarrays 79534.3.1.2 Considerations for the Design of Expression Microarrays 79634.3.1.3 Direct Labeling of RNA for Microarray Hybridization 79634.4 Troubleshooting 799

Acknowledgments 799References 800

35 Experimental RNomics, a Global Approach to Identify Non-codingRNAs in Model Organisms, and RNPomics to Analyze the Non-codingRNP Transcriptome 801Mathieu Rederstorff and Alexander Huttenhofer

35.1 Introduction 80135.2 Computational Analysis of ncRNA Sequences 81135.3 Notes 81235.4 Computational Analysis of ncRNA Sequences 81635.5 Notes 816

Acknowledgments 817References 817

36 Computational Methods for Gene Expression Profiling UsingNext-Generation Sequencing (RNA-Seq) 821John C. Castle

36.1 Introduction 82136.2 Procedure Overview 82236.2.1 Understand the Experiment and the Molecular Biology

Protocol 82336.2.1.1 Library Generation 82336.2.1.2 Sequencing 82536.2.2 Align Reads 82636.2.3 Associate Reads with Transcripts 82736.2.4 Determine Expression and Uncertainty 82836.2.5 Normalization 82836.2.6 Output and Viewing 82836.2.7 Troubleshooting 82936.2.8 The Future Is Bright! 830

XXIV Contents

36.3 Protocols: Useful Algorithms, Formats, and Tools 830References 830

37 Characterization and Prediction of miRNA Targets 833Jean Hausser and Mihaela Zavolan

37.1 Introduction 83337.2 Description 83437.2.1 Building a Set of ‘‘Positives’’ and ‘‘Negatives’’; Obtaining Examples of

Functional and Non-functional miRNA Binding Sites 83537.2.1.1 Comparative genomics 83637.2.1.2 miRNA perturbation and omics 83737.2.1.3 Immunoprecipitation of RISC components 83837.2.1.4 Measuring translation repression directly with polysome

profiles 83937.2.1.5 Which data set should one use for inferring properties that

characterize functional miRNA binding sites? 83937.2.2 Properties of Functional miRNA Binding Sites 84037.2.2.1 The ‘‘seed’’ binding criterion 84037.2.2.2 Evolutionary conservation 84137.2.2.3 Stability of the miRNA–mRNA duplex 84137.2.2.4 Structural accessibility 84137.2.2.5 Sequence composition 84237.2.2.6 Spatial effects 84237.2.3 Combining Properties and Examples into a Predictive Model 84337.2.3.1 Inferring properties that consistently predict miRNA targeting across

data sets 84337.2.3.2 Training a miRNA target prediction model 84637.3 Troubleshooting 84737.3.1 Using miRNA target predictions in an experimental setting 84737.3.1.1 How accurate are miRNA target predictions? 84837.3.1.2 Which miRNA target prediction method should I use? 84937.3.1.3 How many targets does a miRNA have? 85037.3.1.4 Why does a particular high-confidence predicted target not change in

response to miRNA overexpression? 85037.3.1.5 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 85037.3.1.6 The list of targets predicted by method x has a different type of

identifiers (Entrez Gene ID/RefSeq ID/Ensembl transcript/ . . .) thanthe list predicted by method y or the list that one obtains in alarge-scale validation experiment (e.g., microarraymeasurement) 851

37.3.2 The Complexity of Gene Regulation and its Impact on DesigningAccurate miRNA Target Prediction Methods 851References 853

Contents XXV

38 Barcoded cDNA Libraries for miRNA Profiling by Next-GenerationSequencing 861Markus Hafner, Neil Renwick, John Pena, Aleksandra Mihailovic,and Thomas Tuschl

38.1 Introduction 86138.2 Overview of the Method 86238.3 Troubleshooting 872

Acknowledgments 872References 872

39 Transcriptome-Wide Identification of Protein Binding Sites on RNA byPAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking andImmunoprecipitation) 877Jessica I. Hoell, Markus Hafner, 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 87739.2 Troubleshooting 897

Acknowledgments 897References 897

40 Global Analysis of Protein–RNA Interactions with Single-NucleotideResolution Using iCLIP 899Julian Konig, Nicholas J. Mc Glincy, and Jernej Ule

40.1 Introduction 89940.2 Procedure 90040.2.1 Overview 90040.2.2 Antibody and Library Preparation Quality Control 90240.2.3 Oligonucleotide Design 90340.2.4 Troubleshooting 904

Acknowledgments 917References 917

Part IV RNA Function, RNP Analysis, SELEX, RNAi 919

41 Use of RNA Affinity Matrices for the Isolation of RNA BindingProteins 921Markus Englert, Bettina Spath, Steffen Schiffer, Sylvia Rosch,Hildburg Beier, and Anita Marchfelder

41.1 Introduction 92141.2 Applications 92741.2.1 Purification of the Nuclear tRNase Z from Wheat Germ 92741.2.2 Purification of the tRNA-Splicing Ligase from Wheat

Germ 930

XXVI Contents

41.3 Notes 932References 932

42 Biotin-Based Affinity Purification of RNA–Protein Complexes 935Marco Preußner, Silke Schreiner, Inna Grishina, Zsofia Palfi, Jingyi Hui,and Albrecht Bindereif

42.1 Introduction 93542.2 Materials 93742.2.1 Biotinylated Probes 93742.2.2 Affinity Matrices 93742.2.3 Cell Extracts 93842.2.4 Buffers and Solutions 93842.2.5 Additional Materials 93942.3 Methods 93942.3.1 Affinity Purification of RNA–Protein Complexes (RNPs) 93942.3.1.1 Depletion of Total Cell Lysate from SAg-Binding Material

(Preclearing) 94042.3.1.2 Preblocking Streptavidin Agarose Beads 94142.3.1.3 Affinity Selection of RNPs for Biochemical Studies 94142.3.1.4 Elution of Affinity-Selected RNPs for Functional Studies by a

Displacement Oligonucleotide 94542.3.2 Affinity Purification of Specific RNA Binding Proteins by Biotinylated

RNAs 94842.3.3 Depletion of Nuclear Extract with Biotinylated RNA 95142.4 Troubleshooting 95242.4.1 Biotinylated 2′OMe RNA Oligonucleotides 95242.4.2 Extracts and Buffers 95242.4.3 Optimization of the Experimental Conditions, When Yields Are

Low 95242.4.4 Optimization of the Experimental Conditions in the Case of High

Background 953References 953

43 Affinity Purification of Spliceosomal and Small NuclearRibonucleoprotein Complexes 957Julia Dannenberg, Patrizia Fabrizio, Cindy L. Will,and Reinhard Luhrmann

43.1 Introduction 95743.2 Immunoaffinity Purification 95843.2.1 Generation of Antipeptide Antibodies: Peptide Selection Criteria 95843.3 RNA Aptamer-Based Affinity Purification 96343.3.1 Approaches for the Isolation of Native Spliceosomal Complexes 963

Acknowledgments 971References 972

Contents XXVII

44 Study of RNA–Protein Interactions and RNA Structure inRibonucleoprotein Particles (RNPs) 975Virginie Marchand, Annie Mougin, Agnes Mereau, IsabelleBehm-Ansmant, Yuri Motorin, and Christiane Branlant

44.1 Introduction 97544.2 Methods 97844.2.1 RNP Reconstitution 97844.2.1.1 Equipment, Materials, and Reagents 97844.2.1.2 RNA Preparation and Renaturation Step 98044.2.2 EMSA 98144.2.2.1 EMSA Method 98144.2.2.2 Supershift Method 98344.2.2.3 Identification of Proteins Contained in RNP by EMSA Experiments

Coupled to a Second Gel Electrophoresis and Western BlotAnalysis 984

44.2.3 Purification of RNPs Reconstituted in Complex Cellular Extracts 98644.2.4 Methods for RNP Purification Using Tobramycin–Sepharose or

MS2-MBP Affinity Chromatography 98744.2.4.1 Equipment and Materials Common to the Two Approaches 98744.2.4.2 RNP Purification Using Tobramycin–Sepharose 98744.2.4.3 Formation of RNPs in the Cellular Extract 98944.2.4.4 Elution of Purified RNPs under Native Conditions 98944.2.4.5 MS2-MBP Affinity Chromatography 98944.2.4.6 Elution and Analysis of Purified RNPs 99044.2.4.7 Analysis of the Purified RNP Protein Content 99044.2.5 Probing of RNA Structure 99144.2.5.1 Properties of the Probes Used 99144.2.5.2 Equipment, Material, and Reagents 99344.2.5.3 Probing Method 99444.2.6 UV Crosslinking and Immunoselection 99944.2.6.1 Equipment, Materials, and Reagents 100044.2.6.2 UV-Crosslinking Method 100344.3 Commentaries and Pitfalls 100544.3.1 RNP Purification and Reconstitution 100544.3.1.1 RNA Purification and Renaturation 100544.3.1.2 EMSA 100544.3.1.3 Tobramycin–Sepharose Affinity Chromatography 100644.3.2 Probing Conditions 100644.3.2.1 Choice of the Probes Used 100644.3.2.2 Ratio of RNA/Probes 100744.3.3 UV Crosslinking 100844.3.3.1 Photoreactivity of Individual Amino Acids and Nucleotide

Bases 100844.3.3.2 Labeled Nucleotide in RNA 100844.3.4 Immunoprecipitations 1008

XXVIII Contents

44.3.4.1 Efficiency of Immunoadsorbents for Antibody Binding 100844.4 Troubleshooting 100844.4.1 RNP Purification by Tobramycin–Sepharose or MS2-MBP Affinity

Chromatography 100844.4.2 RNP Reconstitution 100944.4.3 RNA Probing 100944.4.4 UV Crosslinking 100944.4.5 Immunoprecipitations 1009

Acknowledgments 1010References 1010

45 Immunopurification of Endogenous RNAs Associated with RNABinding Proteins In vivo 1017Minna-Liisa Anko and Karla M. Neugebauer

45.1 Introduction 101745.2 Description of Methods 101745.2.1 Overview 101745.2.2 Analysis of Coimmunoprecipitated RNA 102245.2.2.1 Microarray Analysis of Immunopurified RNA 102245.2.2.2 RT-PCR Analysis of Immunopurified RNA 102445.2.2.3 Next-Generation Sequencing of Immunopurified RNA 102545.3 Troubleshooting 102545.3.1 Critical Points and Common Problems 102545.3.2 Uncrosslinked or Crosslinked RNA Immunoprecipitation 102645.3.3 Microarray Data Analysis 102645.4 Conclusions 1027

Acknowledgments 1027References 1027

46 Protein–RNA Crosslinking in Native Ribonucleoprotein Particles 1029Olexandr Dybkov, Henning Urlaub, and Reinhard Luhrmann

46.1 Introduction 102946.2 Overall Strategy 103046.3 UV Crosslinking 103146.4 Identification of UV-Induced Protein–RNA Crosslinking Sites by

Primer Extension Analysis 103346.5 Identification of Crosslinked Proteins 103746.6 Troubleshooting 1040

Acknowledgments 1050References 1050

47 Sedimentation Analysis of Ribonucleoprotein Complexes 1055Tanja Rosel, Jan Medenbach, Andrey Damianov, Silke Schreiner, andAlbrecht Bindereif

47.1 Introduction 1055