The second edition of a highly acclaimed handbook and ready reference. Unmatched in its breadth and quality, around 100 specialists from all over the world share their up-to-date expertise and experiences, including hundreds of protocols, complete with explanations, and hitherto unpublished troubleshooting hints. They cover all modern techniques for the handling, analysis and modification of RNAs and their complexes with proteins. Throughout, they bear the practising bench scientist in mind, providing quick and reliable access to a plethora of solutions for practical questions of RNA research, ranging from simple to highly complex. This broad scope allows the treatment of specialized methods side by side with basic biochemical techniques, making the book a real treasure trove for every researcher experimenting with RNA.

Roland K. Hartmann is Professor of Pharmaceutical Chemistry at the Philipps-Universität Marburg (Germany). He studied Biochemistry at the Freie Universität Berlin where in 1988 he received the Ernst Reuter award for outstanding Ph.D. theses. His research interests include natural ribozymes, particularly ribonuclease P, interactions of RNAs with proteins and small ligands, aptamers, antisense and RNA interference.Albrecht Bindereif is Professor of Biochemistry at the University of Giessen (Germany) since 1999. He studied Biochemistry in Tübingen and at the University of California, Berkeley. His postdoctoral work was done in Michael Green's laboratory at Harvard University. His research interests focus on the mechanism and regulation of mRNA splicing, both in the mammalian system and in trypanosomes.Astrid Schön received her PhD from the Institute of Biochemistry at the University of Würzburg, and was a graduate fellow and postdoctoral associate with Dieter Söll at Yale University. She is currently a lecturer at the Institute of Biochemistry, University of Leipzig (Germany). Her research is focused on RNA metabolism and RNA-protein interactions, and the evolution of complex RNA enzymes.Eric Westhof is Professor of Biophysics at the Université Louis Pasteur in Strasbourg (France) since 1988. He studied in Liege and Regensburg and was a Fulbright-Hays Research Fellow at the University of Wisconsin with M. Sundaralingam. His research is centered on the relationships between sequence, structure, evolution and functions of RNA molecules.

Preface XXXV

List of Contributors XXXVII

Part I RNA Synthesis and Detection 1

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

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

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

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

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

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

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

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

Part II Structure Determination 173

9 Direct Determination of RNA Sequence and Modification by Radiolabeling Methods 175Olaf Gimple and Astrid Schön

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

25 Atomic Force Microscopy Imaging and Force Spectroscopy of RNA 527Malte Bussiek, Antonie Schöne, and Wolfgang Nellen

Part III RNA Genomics& Bioinformatics, Global Approaches 547

26 Secondary Structure Prediction 549Gerhard Steger

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

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

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

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

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

32 Molecular Dynamics Simulations of RNA Systems 687Pascal Auffinger

33 Identification and Characterization of Small Non-coding RNAs in Bacteria 719Dimitri Podkaminski, Marie Bouvier, and Jörg Vogel

34 The Identification of Bacterial Non-coding RNAs through Complementary Approaches 787Björn Voß and Wolfgang R. Hess

35 Experimental RNomics, a Global Approach to Identify Non-coding RNAs in Model Organisms, and RNPomics to Analyze the Non-coding RNP Transcriptome 801Mathieu Rederstorff and Alexander Hüttenhofer

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

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

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

39 Transcriptome-Wide Identification of Protein Binding Sites on RNA by PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) 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

40 Global Analysis of ProteinRNA Interactions with Single-Nucleotide Resolution Using iCLIP 899Julian König, Nicholas J. Mc Glincy, and Jernej Ule

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

41 Use of RNA Affinity Matrices for the Isolation of RNA Binding Proteins 921Markus Englert, Bettina Späth, Steffen Schiffer, Sylvia Rösch, Hildburg Beier, and Anita Marchfelder

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

43 Affinity Purification of Spliceosomal and Small Nuclear Ribonucleoprotein Complexes 957Julia Dannenberg, Patrizia Fabrizio, Cindy L. Will, and Reinhard Lührmann

44 Study of RNAProtein Interactions and RNA Structure in Ribonucleoprotein Particles (RNPs) 975Virginie Marchand, Annie Mougin, Agnes Méreau, Isabelle Behm-Ansmant, Yuri Motorin, and Christiane Branlant

45 Immunopurification of Endogenous RNAs Associated with RNA Binding Proteins In vivo 1017Minna-Liisa Änkö and Karla M. Neugebauer

46 ProteinRNA Crosslinking in Native Ribonucleoprotein Particles 1029Olexandr Dybkov, Henning Urlaub, and Reinhard Lührmann

47 Sedimentation Analysis of Ribonucleoprotein Complexes 1055Tanja Rösel, Jan Medenbach, Andrey Damianov, Silke Schreiner, and Albrecht Bindereif

48 Identification and Characterization of RNA Binding Proteins through Three-Hybrid Analysis 1067Felicia Scott and David R. Engelke

49 Experimental Identification of MicroRNA Targets 1087Michaela Beitzinger and Gunter Meister

50 Aptamer Selection against Biological Macromolecules: Proteins and Carbohydrates 1097Franziska Peter and C. Stefan Voertler

51 In Vitro Selection against Small Targets 1139Dirk Eulberg, Christian Maasch, Werner G. Purschke, and Sven Klussmann

52 SELEX Strategies to Identify Antisense and Protein Target Sites in RNA or hnRNP Complexes 1165Martin Lützelberger, Martin R. Jakobsen, and Jørgen Kjems

53 Genomic SELEX 1185Jennifer L. Boots, Katarzyna Matylla-Kulinska, Marek Zywicki, Bob Zimmermann, and Renée Schroeder

54 In vivo SELEX Strategies 1207Thomas A. Cooper

55 Gene Silencing Methods Using Vector-Encoded siRNAs or miRNAs 1221Ying Poi Liu and Ben Berkhout

56 Using Chemical Modification to Enhance siRNA Performance 1243Jesper B. Bramsen, Arnold Grünweller, Roland K. Hartmann, and Jørgen Kjems

Appendix: UV Spectroscopy for the Quantitation of RNA 1279

Index 1283