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. Applications of nucleic acid chemistry are used intensively in material science, as nucleic acid sequences provide a code for information storage and manipulation, as well as the means for self-assembly of complex nanoscale devices and systems. The applications also have their place in the new field of synthetic biology. The pathways by which the nucleoside building blocks of nucleic acids are biosynthesized and incorporated into nucleic acids are important targets for antiviral and chemotherapies. A large variety of modified nucleosides and nucleotides have been designed as tools for studying biochemical processes involving nucleotide-binding proteins (as enzyme substrates or as cofactors), and many more have been evaluated as potential drugs. Detailed protocols for much of the early chemistry can be found in a useful series of books starting with Synthetic Procedures in Nucleic Acid Chemistry, Vols. 1 and 2 (Zorbach and Tipson, 1968) and continuing with four volumes, Nucleic Acid Chemistry: Improved and New Synthetic Procedures, Methods and Techniques, Parts I-IV (Townsend and Tipson, 1978, 1986, 1991). In addition, protocols for the synthesis of oligonucleotides and their analogs and conjugates can be found in two volumes of Methods in Molecular Biology (Agrawal, 1993, 1994). The current methods applied to the synthesis of modified nucleosides and nucleic acids for structure-function studies, as potential therapeutic agents, and as tools for molecular biology, have spawned a unique set of chemistries that provide the fundamental basis on which Current Protocols in Nucleic Acid Chemistry has evolved. Here, the practical aspects of innovative methods for the preparation of modified nucleosides and nucleic acids are strongly emphasized, including N-protection of nucleobases, solid-phase and automated synthesis of oligonucleotides, and conjugation to other molecules. Additional content has been added, including methods for probing DNA and RNA structures, carrying out biophysical and computational analysis, analyzing the binding of nucleic acids to other molecules, creating oligonucleotide arrays, and using nucleosides and RNAi in the fight against infectious diseases and cancers. This publication is available in looseleaf and online formats. For looseleaf purchasers, binders are 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. The material covered in both formats is identical. Subjects in this manual are organized by chapters, 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. For some widely used techniques (such as gel electrophoresis), readers are referred 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 contain 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 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, it is essential to ensure 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 generally subjective and is 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. Contact information for all of the suppliers mentioned in this manual is 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 must be used in strict accordance with local and national regulations. Most of the protocols included in this title are used routinely in the authors' laboratories. These protocols work for them; to make them work for you they have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this protocol collection depends upon readers' observations and suggestions. We encourage readers to send their comments to CurrentProtocols@wiley.com, or to go to any specific unit on our website at http://www.currentprotocols.com, where you can post comments, ask questions, and see author updates. Drs. Donald Bergstrom and Serge Beaucage have retired from our editorial board. They were among the four scientists who took up the challenge to develop this title—the first initiative for Current Protocols in the field of chemistry, a scientific field with a very conservative way of presenting results. We can say now that this was the right decision and that they made a success of it. We thank Don and Serge for their many initiatives and suggestions and, certainly, for their service and contributions as editors. We regret that they have stepped down; we will miss their guidance and wish them success in their new endeavors. The continuing editorial board members take this opportunity to welcome Drs. Martin Egli and Yogesh Sanghvi as new editorial board members. We were very happy that they accepted the invitation to strengthen our board. Martin is a leading scientist in the physicochemistry of nucleic acids, and Yogesh is working at the forefront of the organic synthesis of nucleosides and nucleic acids. Their expertise, experience, and enthusiasm for the field are guarantees for maintaining the high quality of our publications. We will work together as a team to continue serving the nucleoside and nucleic acids community and to take care that our author's contributions will be published in the best way possible. 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 and production staffs at John Wiley & Sons, including Sean Pidgeon, Scott Holmes, Joseph White, Tom Cannon, Allen Ranz, Susan Lieberman, and Sylvia de Hombre. The publisher's commitment and continuing support for a nucleic acid chemistry manual have been essential for realizing and continuing this ambitious project. We are extremely grateful for the critical contributions made by Ann Boyle, who played a key role in initiating the project, and to Beth Harkins, who helped the editorial board develop much of the content. Finally, we gratefully acknowledge the significant roles of past editors Roger Jones and Gary Glick in developing Current Protocols in Nucleic Acid Chemistry during its first half-decade. Overviews of many important topics related to nucleic acid chemistry, structure, and biochemistry. General introduction to fundamental aspects of nucleic acid chemistry. Overview of the design and synthesis of nucleoside-analog antiviral agents. Reviews on many current topics in RNA biochemistry, chemistry, and structure. Includes current laboratory techniques and protocols. Identification of organic compound by mass spectrometry, emphasizing recognition of typical fragmentation patterns for different types of organic compounds. Clear, accessible coverage of modern NMR spectroscopy. methods. Basic biochemistry text that includes fundamentals of nucleic acid biochemistry. The 1999 Oxford Handbook contains the most extensive and comprehensive coverage on nucleic acid structure. Explores the identification of organic compounds by mass spectrometry, infrared spectrometry, and nuclear magnetic resonance spectrometry. Laboratory manual that describes very basic techniques for synthetic chemistry.