Douglas T. Gjerde, Lee Hoang, and David Hornby RNA Purifi cation and Analysis Further Reading J.S. Fritz, D.T. Gjerde L.W. Miller (Ed.) Ion Chromatography Probes and Tags to Study Biomolecular Function 2009 ISBN: 978-3-527-32052-3 for Proteins, RNA, and Membranes 2008 ISBN: 978-3-527-31566-6 G. Carta, A. Jungbauer Protein Chromatography J. von Hagen (Ed.) Process Development and Scale-Up Proteomics Sample Preparation 2009 ISBN: 978-3-527-31819-3 2008 ISBN: 978-3-527-31796-7 P. Herdewijn (Ed.) R.K. Hartmann, A. Bindereif, A. Schön, Modifi ed Nucleosides E. Westhof (Eds.) in Biochemistry, Biotechnology and Medicine Handbook of RNA Biochemistry 2008 2005 ISBN: 978-3-527-31820-9 ISBN: 978-3-527-30826-2 Douglas T. Gjerde, Lee Hoang, and David Hornby RNA Purifi cation and Analysis Sample Preparation, Extraction, Chromatography The Authors All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and Dr. Douglas T. Gjerde publisher do not warrant the information contained PhyNexus, Inc. in these books, including this book, to be free of 3670, Charter Park Drive errors. Readers are advised to keep in mind that San José, CA 95136 statements, data, illustrations, procedural details or USA other items may inadvertently be inaccurate. Dr. Lee Hoang Library of Congress Card No.: applied for PhyNexus, Inc. 3670, Charter Park Drive British Library Cataloguing-in-Publication Data San José, CA 95136 A catalogue record for this book is available from USA the British Library. Dr. David Peter Joseph Hornby Bibliographic information published by University of Sheffi eld the Deutsche Nationalbibliothek Department of Molecular Biology The Deutsche Nationalbibliothek lists this Firth Court, Western Bank publication in the Deutsche Nationalbibliografi e; Sheffi eld S10 2TN detailed bibliographic data are available on the United Kingdom Internet at http://dnb.d-nb.de © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfi lm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifi cally marked as such, are not to be considered unprotected by law. Printed in the Federal Republic of Germany Printed on acid-free paper Cover design Schulz Grafi k-Design, Fußgönheim Typesetting SNP Best-set Typesetter Ltd., Hong Kong Printing betz-druck GmbH, Darmstadt Bookbinding Litges & Dopf Buchbinderei GmbH, Heppenheim ISBN: 978-3-527-32116-2 V Contents Preface IX Acknowledgments XI 1 RNA Extraction, Separation, and Analysis 1 1.1 The Need to Be Able to Extract, Manipulate, and Analyze RNA 1 1.2 Using Chemical Tools to Solve the Problem of Analysis of Biological Processes 3 1.3 The Principle of Chromatography and Solid-Phase Extraction 4 1.3.1 Principle of Chromatography 4 1.3.2 Mobile Phase Gradient Controls Elution 5 1.3.3 Different Types of Column and Eluent Chemistries 6 1.3.4 The Principle of Solid-Phase Extraction 8 1.4 RNA Chromatography 10 1.5 Enzymatic Treatment of RNA and Analysis 13 1.5.1 Polyacrylamide Gel Electrophoresis 14 1.5.2 RNA Structure Probing with Ribonuclease Enzymes 14 1.6 Content and Organization of This Book 15 References 16 2 Biological and Chemical RNA 17 2.1 Why Classify RNA with Biology and Chemistry? 17 2.1.1 Chemical Classifi cation of RNA 18 2.1.2 Biological Classifi cation of RNA 19 2.2 Prokaryotic Cellular RNA 20 2.3 Prokaryote Sample Type 23 2.3.1 Escherichia coli 23 2.3.2 Other Bacteria 24 2.4 Eukaryotic Cellular RNA 24 2.5 Eukaryote Sample Type 27 2.5.1 Yeast 29 2.5.2 Other Fungi 30 2.5.3 Simple Multicellular Organism 30 RNA Purifi cation and Analysis: Sample Preparation, Extraction, Chromatography Douglas T. Gjerde, Lee Hoang, and David Hornby Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32116-2 VI Contents 2.5.4 Soft Animal 30 2.5.5 Hard Animal 31 2.5.6 Plant 31 2.5.7 Cell Culture 31 2.6 Other Samples 32 2.6.1 Virus 32 2.6.2 Soil and Rock 32 2.7 Synthetic RNA 32 2.7.1 Aptamers 33 2.7.2 SELEX 34 2.7.3 Short Hairpin RNAs 34 References 34 3 RNA Separation: Substrates, Functional Groups, Mechanisms, and Control 37 3.1 Solid-Phase Interaction 37 3.1.1 Adsorption of Sample Compounds and Sample Matrix Compounds 37 3.1.2 Roles of Solid-Phase Substrate and Functional Group 39 3.1.3 Correlation of Interaction Type, Functional Group, and Substrate 40 3.1.4 RNA Structure and Solid Surface Interaction 41 3.2 The Solid-Phase Substrate and Attachment of Functional Groups 43 3.2.1 Polymeric Resin Substrates 44 3.2.2 Porous and Nonporous Polymeric Resins 45 3.2.3 Monolith Polymeric Columns 47 3.2.4 Functionalization of the Polymer 48 3.2.5 Silica–Glass-Based Substrates 50 3.2.6 Functionalization of Silica 51 3.2.7 Agarose and Cellulose Affi nity Substrates 53 3.2.8 Dextran and Polyacrylamide Gel Filtration Substrates 53 3.3 Reverse-Phase Ion-Pairing Separation Mechanism 54 3.4 Ion-Exchange Separation Mechanism 57 3.5 Chaotropic Denaturing Interaction Mechanism 61 3.6 Hybridization 62 3.6.1 SELEX 62 3.7 Gel Filtration 63 References 64 4 RNA Extraction and Analysis 67 4.1 Transcription 67 4.1.1 RNA Catalysis 69 4.1.2 RNA–Protein Complex Interactions 69 4.1.3 Pre-mRNA Splicing 71 4.2 Translation 72 Contents VII 4.2.1 Post-Transcriptional Control of Eukaryotic Gene Expression 76 4.3 Gene Regulation 76 4.3.1 RNA Interference Pathway 76 4.3.2 Micro RNAs and Their Role in Gene Regulation 78 4.4 Use of siRNA to Investigate Gene Function 79 References 79 5 RNA Chromatography 81 5.1 Development of RNA Chromatography 81 5.2 RNA Chromatography Instrumentation 85 5.2.1 The Column Oven 85 5.2.2 Ultraviolet (UV) and Fluorescence Detection 86 5.2.3 Fragment Collection 86 5.3 RNA Chromatography Conditions 87 5.4 Temperature Modes of RNA Chromatography 88 5.4.1 Nondenaturing Mode 89 5.4.2 Partially Denaturing Mode 89 5.4.3 Fully Denaturing Mode 90 5.5 Comparison of Gel Electrophoresis and Liquid Chromatography 90 5.5.1 Gel Electrophoresis 90 5.5.2 Liquid Chromatography 93 5.6 Analysis of Human Telomerase RNA Under Nondenaturing Conditions 95 References 98 6 RNA Chromatography Separation and Analysis 101 6.1 Features of RNA Chromatography 101 6.2 Separation of Double-Stranded and Single-Stranded RNA 102 6.3 Separation of Cellular RNA Species 107 6.4 Separation of Messenger RNA from Total and Ribosomal RNA 108 6.5 Analysis of Transfer RNA 109 6.6 Chromatography and Analysis of Synthetic Oligoribonucleotides 111 6.7 Application of RNA and DNA Chromatography in cDNA Library Synthesis 114 6.8 DNA Chromatography Analyses of RT-PCR and Competitive RT-PCR Products 117 6.9 Alternative Splicing 120 6.10 Differential Messenger RNA Display via DNA Chromatography 121 References 124 7 RNA Structure–Function Probing 127 7.1 Defi nition of the Structure–Function Paradigm 127 7.1.1 Francis Crick, and Predicting the Existence of tRNA 130 7.1.2 The Discovery of Ribozymes 133 7.2 Structure Determination of RNA 135 VIII Contents 7.2.1 Primary Structure 136 7.2.2 Secondary Structure 136 7.2.3 Tertiary Structure 137 7.2.4 Quaternary Structure 138 7.3 Footprinting, Model Building, and Functional Investigations 139 7.3.1 Chemical Probing and Cleavage 140 7.3.2 Modifi cation Interference 143 7.3.3 FRET 144 References 144 Appendix 1 Chromatographic Separation Equations and Principles for RNA Separation 147 Appendix 2 HPLC Instrumentation and Operation 159 Appendix 3 RNA Chromatographic System Cleaning and Passivation Treatment 185 Index 191 IX Preface Unlocking the role of RNA in biological cellular processes has proved to be more challenging than perhaps many investigators had fi rst believed would be the case. However, it is also becoming apparent that, as the mysteries of RNA function are revealed, greater rewards will be gained than had been imagined. While life is indeed complicated, this book takes an approach to understanding life by examin- ing it as a set of discrete, simple chemical reactions – the control of which generates the complexity. In continuing with this theme of reductionism, RNA is particularly amenable to in vitro analysis and, by isolating the RNA and asking questions in a very controlled manner, the details of specifi cs relating to the catalysis, affi nities and mechanisms of RNA can be identifi ed. As a result, major advances should be achieved in our fundamental understanding of biology, and perhaps even greater medical rewards can be gained. RNA is fragile and complex in both its structure and function, and advanced tools are required for the reliable capture, purifi cation and analysis of its various types. It is not the purpose of this book to provide a series of “ cookbook ” proce- dures for RNA ’ s extraction, separation, and analysis; rather, it is intended as a “ tool book ” , in which we describe the chemical principles that can be used as the foundations for the various methods and tools used to investigate RNA. For that reason, some examples are provided to illustrate the various concepts. By under- standing these basic chemical concepts, and how to apply them in terms of the way in which RNA can be manipulated, the biologist should be able to better use the routine products and methods that are currently available, or perhaps develop new techniques for coping with any new - found problems associated with RNA. In this book, we have brought together the concepts of both biology and chem- istry to describe what these tools are, and how they work. Some of the tools described, such as spin columns and precipitation procedures, will already be familiar to the biologist, as will the various procedures described for the different types of RNA. Some other tools may be unfamiliar to the reader, however, and consequently the potential of high - performance liquid chromatography is described in detail, as are the basic instrumentation, the separation columns, and the means by which these separations are controlled. RNA Purifi cation and Analysis: Sample Preparation, Extraction, Chromatography Douglas T. Gjerde, Lee Hoang, and David Hornby Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32116-2 X Preface The special instrumentation and columns required for RNA separations have led to this technology being referred to as “ RNA Chromatography ” , with the breadth of the subject being illustrated clearly by the many applications described. So, we hope that this book will be used by biologists not only as a means of expanding their arsenal of tools for RNA - based investigations, but also to help them use these tools to great effect. With such techniques available it should, in time, be possible to solve – in creative manner – the vast array of problems encoun- tered in RNA - related research. May 2009 D ouglas T. Gjerde Lee Hoang David Hornby