Preface

Abstract

Chemistry has played a pivotal role in the development of molecular biology and biotechnology. The value of synthetic oligonucleotides became apparent when they provided the means for deciphering the genetic code. Modern cloning techniques and nucleic acid sequencing depend on the ability to obtain synthetic primers. In recent years intensive research has driven the technological development of high-throughput methods for nucleic acid analysis. Moreover, modified nucleic acids have been extensively studied and identified as highly specific therapeutic agents. At the same time, research efforts have provided new means to probe the structure of nucleic acids for a better understanding of all aspects of nucleic acid function and interactions in biology. Until recently, nucleic acid chemistry has focused on the synthesis of modified nucleosides and nucleotides as potential drugs and as tools for studying biochemical processes involving nucleotide binding proteins (as enzyme substrates or as cofactors). Detailed protocols for much of this chemistry can be found in a useful series of books starting with Synthetic Procedures in Nucleic Acid Chemistry, Vols. 1 and 2 (W.W. Zorbach and R. Stuart Tipson, eds., Interscience Publishers, New York, 1968) and continuing with four volumes, Nucleic Acid Chemistry: Improved and New Synthetic Procedures, Methods and Techniques, Parts I-IV, (Leroy B. Townsend and R. Stuart Tipson, eds., John Wiley and Sons, New York, parts I and II, 1978, part III, 1986, and part IV, 1991). The current methods applied to the synthesis of modified nucleic acids for structure-function studies, potential therapeutic agents, and as tools for molecular biology, spawned a unique set of chemistries that provide the fundamental basis on which this volume of Current Protocols has evolved. In Current Protocols in Nucleic Acid Chemistry, the practical aspects of innovative methods for the preparation of modified nucleic acids are strongly emphasized. Although a number of useful literature overviews are included in the volume, authors were requested to comply with editorial policy and limit the number of reference citations. The first four chapters describe detailed methodology for the synthesis of both natural and modified oligonucleotides. Procedures for the analysis and characterization of oligonucleotides are presented in Chapters 7 and 10. Other chapters focus on chemical and physical methods for probing the structure and function of nucleic acids. Separate chapters have been devoted to important new areas of nucleic acid chemistry including combinatorial methods and RNA folding. As nucleic acid chemistry continues to evolve, new and improved techniques relevant to all aspects of the discipline will be incorporated into Current Protocols in Nucleic Acid Chemistry. This publication is available in looseleaf, CD-ROM, and Intranet formats. For looseleaf purchasers, a binder is provided to accommodate the growth of the manual via the quarterly update service. The looseleaf format of the binder allows easy insertion of new pages, units, and chapters that are added. The index and table of contents are updated with each supplement. Purchasers of the CD-ROM and Intranet versions receive a completely new disc every quarter and should dispose of their outdated discs. The material covered in all versions is identical. Subjects in this manual are organized by chapters and sections, and protocols are contained in units. Units generally describe a method and include one or more protocols with listings of materials, steps and annotations, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the method; there are also “overview” units containing theoretical discussions that lay the foundation for subsequent protocols. Page numbering in the looseleaf version reflects the modular arrangement by unit; for example, page 2.3.5 refers to Chapter 2 (Protection of Nucleosides for Oligonucleotide Synthesis), UNIT Unavailable (Protection of the 5′-Hydroxy Function of Nucleosides), page 5 of that particular unit. Many reagents and procedures are employed repeatedly throughout the manual. Instead of duplicating this information, cross-references among units are used extensively. Cross-referencing helps to ensure that lengthy and complex protocols are not overburdened with steps describing auxiliary procedures needed to prepare raw materials and analyze results. Certain units that describe commonly used techniques and recipes are cross-referenced in other units that describe their application. For some widely used techniques (such as gel electrophoresis), readers are cross-referenced to APPENDIX Unavailable. Because this publication is first and foremost a compilation of laboratory techniques in nucleic acid chemistry, we have not offered extensive instructive material. We have, however, included explanatory information where required to help readers gain an intuitive grasp of the procedures. Some chapters begin with special overview units that describe the state of the art of the topic matter and provide a context for the procedures that follow. Chapter and unit introductions describe how the protocols that follow connect to one another, and annotations to the actual protocol steps describe what is happening as a procedure is carried out. Finally, the Commentary that closes each protocol unit describes background information regarding the historical and theoretical development of the method, as well as alternative approaches, critical parameters, troubleshooting guidelines, anticipated results, and time considerations. All units contain cited references and many indicate key references to inform users of particularly useful background reading, original descriptions, or applications of a technique. Many units in the manual contain groups of protocols, each presented with a series of steps. The basic protocol is presented first in each unit and is generally the recommended or most universally applicable approach. Alternate protocols are given where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a variation in approach, or where requirements for the end product differ from those in the basic protocol. Support protocols describe additional steps that are required to perform the basic or alternate protocols; these steps are separated from the core protocol because they might be applicable to other uses in the manual, or because they are performed in a time frame separate from the basic protocol steps. Reagents required for a protocol are itemized in the materials list before the procedure begins. Many are common stock solutions, others are commonly used buffers or media, whereas others are solutions unique to a particular protocol. Recipes for the latter solutions are supplied in each unit, following the protocols (and before the commentary) under the heading Reagents and Solutions. It is important to note that the names of some of these special solutions might be similar from unit to unit (e.g., SDS sample buffer) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used stock solutions and buffers are listed once in APPENDIX Unavailable. These universal recipes are cross-referenced parenthetically in the materials lists rather than repeated with every usage. In some instances throughout the manual, we have recommended commercial suppliers of chemicals, biological materials, or equipment. This has been avoided wherever possible, because preference for a specific brand is subjective and is generally not based on extensive comparison testing. Our guidelines for recommending a supplier are that (1) the particular brand has actually been found to be of superior quality, or (2) the item is difficult to find in the marketplace. The purity of chemical reagents frequently varies with supplier. Generally reagent grade chemicals are preferred. Special care must be paid to procedures that require dry solvents. Different suppliers provide special anhydrous grade solvents which may vary in water content depending on the supplier. Addresses, phone numbers, and facsimile numbers of all suppliers mentioned in this manual are provided in the SUPPLIERS APPENDIX. Anyone carrying out these protocols will encounter the following hazardous or potentially hazardous materials: (1) radioactive substances, (2) toxic chemicals and carcinogenic or teratogenic reagents, (3) pathogenic and infectious biological agents, and (4) recombinant DNA. Most governments regulate the use of these materials; it is essential that they be used in strict accordance with local and national regulations. Cautionary notes are included in many instances throughout the manual, but we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials be used in strict accordance with local and national regulations. Most of the protocols included in this manual are used routinely in our own laboratories. These protocols work for us; to make them work for you we have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this manual depends upon readers' observations and suggestions. Consequently, a self-mailing reader-response survey can be found at the back of the manual (and is included with each supplement); we encourage readers to send in their comments. This manual is the product of dedicated efforts by many of our scientific colleagues who are acknowledged in each unit and by the hard work of the Current Protocols editorial staff at John Wiley and Sons. The publisher's commitment and continuing support for a nucleic acid chemistry manual were essential for realizing this ambitious project. We are extremely grateful for the critical contributions by Ann Boyle (Series Editor) who kept the editors and the contributors on track and played a key role in bringing the entire project to completion. Other skilled members of the Current Protocols staff who contributed to the project include Joseph White, Kathy Wisch, Tom Cannon Jr., Alice Ro, Alda Trabucchi, Michael Gates, and Liana Scalettar. The extensive copyediting required to produce an accurate protocols manual was ably handled by Rebecca Barr, Allen Ranz, Ben Gutman, Beth Harkins, Monte Kendrick, and Candace Levy. General introduction to fundamental aspects of nucleic acid chemistry. An overview of many important topics related to nucleic acid chemistry, structure, and biochemistry. Basic biochemistry text which includes fundamentals of nucleic acid biochemistry. Comprehensive overview of everything you would ever want to know about nucleic acid structure. Reviews on many current topics in RNA biochemistry, chemistry and structure. Laboratory manual that describes very basic techniques for synthetic chemistry.