Cover -- Title Page -- Copyright -- Contents -- Preface -- List of Contributors -- Part I RNA Synthesis and Detection -- Chapter 1 Enzymatic RNA Synthesis Using Bacteriophage T7 RNA Polymerase -- 1.1 Introduction -- 1.2 Description of Method - T7 Transcription In vitro -- 1.2.1 Templates -- 1.2.1.1 Strategy (i): Insertion into a Plasmid -- 1.2.1.2 Strategy (ii): Direct Use of Templates Generated by PCR -- 1.2.1.3 Strategy (iii): Annealing of a T7 Promoter DNA Oligonucleotide to a Single-Stranded Template -- 1.2.2 Special Demands on the RNA Product -- 1.2.2.1 Homogeneous 5' and 3' Ends, Small RNAs, Functional Groups at the 5' End -- 1.2.2.2 Modified Substrates -- 1.3 Transcription Protocols -- 1.3.1 Transcription with Unmodified Nucleotides -- 1.3.2 Transcription with 2' -Fluoro-Modified Nucleotides -- 1.3.3 T7 Transcripts with 5' - Cap Structures -- 1.3.4 Purification -- 1.4 Troubleshooting -- 1.4.1 Low or No Product Yield -- 1.5 Rapid Preparation of T7 RNA Polymerase -- 1.5.1 Required Material -- 1.5.1.1 Medium -- 1.5.1.2 Buffers and Solutions -- 1.5.1.3 Electrophoresis and Chromatography -- 1.5.2 Procedure -- 1.5.2.1 Cell Growth, Induction, and Test for Expression of T7 RNAP -- 1.5.2.2 Purification of T7 RNAP -- 1.5.3 Notes and Troubleshooting -- References -- Chapter 2 Production of RNAs with Homogeneous 5' - and 3' -Ends -- 2.1 Introduction -- 2.2 Description of Approach -- 2.2.1 Cis-Cleaving Autocatalytic Ribozyme Cassettes -- 2.2.1.1 The 5' -Cassette -- 2.2.1.2 The 3' -Cassette -- 2.2.1.3 Purification of Released RNA Product and Conversion of End Groups -- 2.2.2 Trans-Cleaving Ribozymes for the Generation of Homogeneous 3' Ends -- 2.2.3 Further Strategies toward Homogeneous Ends -- 2.3 Critical Experimental Steps, Changeable Parameters, Troubleshooting.

2.3.1 Construction of Cis-Cleaving 5'- and 3'-Cassettes -- 2.4 PCR Protocols -- 2.5 Potential Problems -- References -- Chapter 3 RNA Ligation -- 3.1 General Introduction -- 3.1.1 T4 Polynucleotide Ligases -- 3.1.2 Reaction Mechanism -- 3.1.3 Advantages of T4 DNA Ligase for RNA Ligation -- 3.1.4 Chapter Structure -- 3.2 RNA Ligation Using T4 DNA Ligase (T4 Dnl) -- 3.2.1 Overview of the RNA Ligation Method Using the T4 DNA Ligase (T4 Dnl) -- 3.2.2 Large-Scale Transcription and Purification of RNAs -- 3.2.3 Generating Homogeneous Acceptor 3' -Ends for Ligation -- 3.2.4 Site-Directed Cleavage with RNase H -- 3.2.5 Dephosphorylation and Phosphorylation of RNAs -- 3.2.6 RNA Ligation -- 3.2.7 Troubleshooting -- 3.3 Simultaneous Splint Ligation of Five RNA Fragments to Generate RNAs for FRET Experiments -- 3.3.1 Introduction -- 3.3.2 Construct Design -- 3.3.3 Troubleshooting -- 3.3.3.1 Low Overall Ligation Efficiency -- 3.3.3.2 Undesired Ligation By-products -- 3.3.3.3 RNA Degradation -- 3.4 T4 RNA Ligase(s) -- 3.4.1 Introduction -- 3.4.2 Mechanism and Substrate Specificity -- 3.4.2.1 Early Studies -- 3.4.2.2 Substrate Specificity and Reaction Conditions -- 3.4.3 Applications of T4 RNA Ligase -- 3.4.3.1 End-Labeling -- 3.4.3.2 Circularization -- 3.4.3.3 Intermolecular Ligation of Polynucleotides -- 3.4.4 T4 RNA Ligation of Large RNA Molecules -- 3.4.5 Application Examples and Protocols -- 3.4.5.1 Production of Full-Length tRNAs -- 3.4.6 Troubleshooting -- References -- Chapter 4 Northern Blot Detection of Small RNAs -- 4.1 Introduction -- 4.1.1 Isolation of RNA -- 4.1.1.1 Kits -- 4.1.1.2 Do it Yourself -- 4.1.1.3 Quality Control -- 4.1.2 Native versus Denaturing Gels -- 4.1.3 Transfer of RNA and Fixation to Membranes -- 4.1.4 Hybridization with a Complementary Probe -- 4.1.4.1 Design of DNA/LNA Mixmer Probes.

4.1.5 Detection of DIG-Labeled Probes -- 4.1.6 Troubleshooting -- 4.1.7 Application Example -- 4.1.8 Limitations of the Method -- 4.2 Northern Hybridization Protocols -- References -- Chapter 5 Rapid, Non-Denaturing, Large-Scale Purification of In Vitro Transcribed RNA Using Weak Anion-Exchange Chromatography -- 5.1 Introduction -- 5.2 Materials -- 5.2.1 Cloning and Plasmid Purification -- 5.2.2 In Vitro Transcription -- 5.2.3 Weak Anion-Exchange FPLC -- 5.3 Protocols for Plasmid Design and Preparation, RNA Transcription, and Weak Anion-Exchange Purification -- 5.4 Troubleshooting -- Acknowledgments -- References -- Chapter 6 3' -Terminal Attachment of Fluorescent Dyes and Biotin -- 6.1 Introduction -- 6.2 Description of Method -- 6.3 History of the Method -- 6.4 Troubleshooting -- 6.4.1 Problems Caused Before the Labeling Reaction -- 6.4.1.1 Quality of the RNA 3' Ends -- 6.4.1.2 Purity of the RNA to Be Labeled -- 6.4.2 Problems with the Labeling Reaction Itself -- 6.4.2.1 pH of Reagents -- 6.4.2.2 Stability of Reagents -- 6.4.3 Postlabeling Problems -- 6.4.3.1 Removal of Labeling Reagents -- 6.4.3.2 Loss of RNA Material during Downstream Purification -- 6.4.3.3 Stability of Labeled RNA -- Acknowledgment -- References -- Chapter 7 Chemical RNA Synthesis, Purification, and Analysis -- 7.1 Introduction -- 7.2 Description -- 7.2.1 The Solid-Phase Synthesis of RNA -- 7.2.2 Deprotection -- 7.2.3 Purification -- 7.2.3.1 Anion-Exchange HPLC Purification -- 7.2.3.2 Reversed-Phase HPLC Purification of Trityl-On RNA -- 7.2.3.3 Detritylation of Trityl-On RNA -- 7.2.3.4 Desalting by HPLC -- 7.2.4 Analysis of the Purified RNA -- 7.3 Troubleshooting -- References -- Chapter 8 Modified RNAs as Tools in RNA Biochemistry -- 8.1 Introduction -- 8.1.1 Modification Strategy: the Phosphoramidite Method.

8.1.2 Modification Strategy: Postsynthetic Labeling -- 8.2 Description of Methods -- 8.2.1 Postsynthetic Modification: the 2' -Amino Approach -- 8.2.2 Reaction of 2' -Amino Groups with Succinimidyl Esters -- 8.2.3 Reaction of 2' -Amino Groups with Aromatic Isothiocyanates -- 8.2.4 Reaction of 2' -Amino Groups with Aliphatic Isocyanates -- 8.3 Experimental Protocols -- 8.3.1 Synthesis of Aromatic Isothiocyanates and Aliphatic Isocyanates -- 8.3.2 Postsynthetic Labeling of 2' -Amino-Modified RNA -- 8.3.3 Postsynthetic Labeling of 4-Thiouridine-Modified RNA -- 8.3.4 Verification of Label Incorporation -- 8.3.5 Potential Problems and Troubleshooting -- References -- Part II Structure Determination -- Chapter 9 Direct Determination of RNA Sequence and Modification by Radiolabeling Methods -- 9.1 Introduction -- 9.2 General Methods -- 9.3 Isolation of Pure RNA Species from Biological Material -- 9.3.1 Preparation of Size-Fractionated RNA -- 9.3.2 Isolation of a Single Unknown RNA Species Following a Functional Assay -- 9.3.2.1 Solutions for Electrophoresis, Staining, and Elution of RNAs from Gels -- 9.3.2.2 Two-Dimensional Electrophoresis of RNA -- 9.3.2.3 Comments on the Electrophoretic Purification and Elution of RNA Species -- 9.3.3 Isolation of Single RNA Species with Partially Known Sequence -- 9.3.3.1 Materials for Hybrid Selection of Single RNA Species -- 9.4 Radioactive Labeling of RNA Termini -- 9.4.1 Materials for 5' -End Labeling of RNAs -- 9.4.2 3' -Labeling of RNAs -- 9.4.2.1 Materials for 3' -End Labeling of RNAs -- 9.5 Sequencing of End-Labeled RNA -- 9.5.1 Sequencing by Base-Specific Enzymatic Hydrolysis of End-Labeled RNA -- 9.5.1.1 Materials Required for Enzymatic Sequencing -- 9.5.1.2 Interpretation and Troubleshooting.

9.5.2 Sequencing by Base-Specific Chemical Modification and Cleavage -- 9.5.2.1 Materials Required for Chemical Sequencing -- 9.5.2.2 Interpretation and Troubleshooting -- 9.6 Determination of Terminal RNA Sequences by Two-dimensional Mobility Shift -- 9.6.1 Materials Required for Mobility Shift Analysis -- 9.7 Determination of Modified Nucleotides by Postlabeling Methods -- 9.7.1 Analysis of Total Nucleotide Content -- 9.7.1.1 Materials Required for RNA Nucleotide Analysis -- 9.7.1.2 Interpretation and Troubleshooting -- 9.7.2 Determination of Position and Identity of Modified Nucleotides -- 9.7.2.1 Interpretation and Troubleshooting -- 9.8 Conclusions and Outlook -- Acknowledgments -- References -- Chapter 10 Probing RNA Structure In Vitro with Enzymes and Chemicals -- 10.1 Introduction -- 10.2 Enzymatic and Chemical Probes -- 10.2.1 Enzymes -- 10.2.2 Base-Specific Chemical Probes -- 10.2.3 Backbone-Specific Chemical Probes -- 10.3 In Vivo DMS Modification -- 10.3.1 Generalities -- 10.3.2 In Vivo Probing -- 10.4 Commentary -- 10.4.1 Critical Parameters -- 10.4.1.1 RNA Preparation -- 10.4.1.2 Homogeneous RNA Conformation -- 10.4.1.3 Chemical and Enzymatic Probing -- 10.4.1.4 In Vivo DMS Mapping -- 10.5 Troubleshooting -- Acknowledgments -- References -- Chapter 11 Probing RNA Solution Structure by Photocrosslinking: Incorporation of Photoreactive Groups at RNA Termini and Determination of Crosslinked Sites by Primer Extension -- 11.1 Introduction -- 11.1.1 Applications of RNA Modifications -- 11.1.2 Techniques for the Incorporation of Modified Nucleotides -- 11.2 Description -- 11.2.1 5' -End Modification by Transcription Priming.

11.2.2 Chemical Phosphorylation of Nucleosides to Generate 5' -Monophosphate or 5' -Monophosphorothioate Derivatives.