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Leading researchers summarize and describe their experience and techniques for synthesizing oligonucleotides and their analogs. The protocols presented cover all DNA analogs that have been studied to date and constitute the single most extensive source of knowledge about DNA chemistry available.
A collection of powerful new techniques for oligonucleotide synthesis and for the use of modified oligonucleotides in biotechnology. Among the protocol highlights are a novel two-step process that yields a high purity, less costly, DNA, the synthesis of phosphorothioates using new sulfur transfer agents, the synthesis of LNA, peptide conjugation methods to improve cellular delivery and cell-specific targeting, and triple helix formation. The applications include using molecular beacons to monitor the PCR amplification process, nuclease footprinting to study the sequence-selective binding of small molecules of DNA, nucleic acid libraries, and the use of small interference RNA (siRNA) as an inhibitor of gene expression.
With contributions from leading experts, this book is the first to focus solely on addressing the problems and reviewing the strategies currently being used to improve the delivery of antisense nucleic acids. Important delivery issues, such as improving biological stability, improving cell-specific targeting and cellular uptake, manipulating subcellular distribution and producing liposomal delivery systems for antisense agents are comprehensively covered in this volume. This book links review-type articles with contributions that contain exciting never-before-published data on the cellular delivery of oligonucleotides. It stimulates reading for both established researchers and newcomers to the antisense field.
This book presents the latest knowledge on a broad range of topics relating to the synthesis of natural and artificial oligonucleotides with therapeutic potential. Nucleic acid-based therapeutics are attracting much attention, and numerous therapeutic oligonucleotides, such as antisense oligonucleotides, siRNAs, splice-switching oligonucleotides, and nucleic acid aptamers, are being evaluated in clinical trials for the treatment of a variety of diseases. Synthesis of Therapeutic Oligonucleotides covers a broad range of topics in the field that are of high relevance to researchers, including the synthesis of natural and chemically modified oligonucleotides, the development of novel nucleic acid analogs, industrial scale synthesis and purification of oligonucleotides, and important aspects of chemistry, manufacturing, and controls (CMC). The aim is to provide new insights and inspire fresh ideas in nucleic acid chemistry that may ultimately lead to novel concepts and techniques and the discovery of more effective nucleic acid drugs. The book will be of high value for both established researchers in the field and students intending to specialize in nucleic acid chemistry research.
When first conceived, not only was the aim of Protocols for Oligo nucleotides and Analogs to provide wide coverage of the ohgonuc- otide chemistry field for readers who are well versed within the field, but also to give investigators just entering into the field a new perspective. The very first book on this topic was edited and published by Michael Gait in 1984, in whose laboratory I encountered the newer aspects of oligonucleotide chemistry. Since then, oligonucleotide research has developed to such an extent that its uses extend far beyond basic studies, and now find wide application throughout clinical science as well. Until recently, the major application of oligonucleotides has been in the area of DNA-based diagnostic and "antisense oligonucleotid- based therapeutic approaches. However, oligonucleotides are now also being used as therapeutic agents and are thus frequently found in clinical trials in humans. Synthesis of unmodified oligonucleotides using automated synthe sizers has become a common practice in numerous laboratories. How ever, improvements on the existing techniques and the introduction of ever newer methods for oligonucleotide synthesis is constantly driving ahead in the leading research laboratories. And several new oligonucle otide analogs have been synthesized and studied for their individual prop erties in recent years. The present volume strives to bring the readers the most up-to-date information on the newest aspects of synthesis of oligo nucleotides and their analogs. A separate volume covers synthesis of oligonucleotide conjugates, along with most of the analytical techniques presently used for analysis of oligonucleotides.
A comprehensive review of contemporary antisense oligonucleotides drugs and therapeutic principles, methods, applications, and research Oligonucleotide-based drugs, in particular antisense oligonucleotides, are part of a growing number of pharmaceutical and biotech programs progressing to treat a wide range of indications including cancer, cardiovascular, neurodegenerative, neuromuscular, and respiratory diseases, as well as other severe and rare diseases. Reviewing fundamentals and offering guidelines for drug discovery and development, this book is a practical guide covering all key aspects of this increasingly popular area of pharmacology and biotech and pharma research, from the basic science behind antisense oligonucleotides chemistry, toxicology, manufacturing, to safety assessments, the design of therapeutic protocols, to clinical experience. Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. While the idea of antisense oligonucleotides to target single genes dates back to the 1970's, most advances have taken place in recent years. The increasing number of antisense oligonucleotide programs in clinical development is a testament to the progress and understanding of pharmacologic, pharmacokinetic, and toxicologic properties as well as improvement in the delivery of oligonucleotides. This valuable book reviews the fundamentals of oligonucleotides, with a focus on antisense oligonucleotide drugs, and reports on the latest research underway worldwide. • Helps readers understand antisense molecules and their targets, biochemistry, and toxicity mechanisms, roles in disease, and applications for safety and therapeutics • Examines the principles, practices, and tools for scientists in both pre-clinical and clinical settings and how to apply them to antisense oligonucleotides • Provides guidelines for scientists in drug design and discovery to help improve efficiency, assessment, and the success of drug candidates • Includes interdisciplinary perspectives, from academia, industry, regulatory and from the fields of pharmacology, toxicology, biology, and medicinal chemistry Oligonucleotide-Based Drugs and Therapeutics belongs on the reference shelves of chemists, pharmaceutical scientists, chemical biologists, toxicologists and other scientists working in the pharmaceutical and biotechnology industries. It will also be a valuable resource for regulatory specialists and safety assessment professionals and an important reference for academic researchers and post-graduates interested in therapeutics, antisense therapy, and oligonucleotides.
This volume in the Methods in Molecular Biology series presents established and emerging methods for the application of oligonucleotides as therapeutics. Includes protocols for optimizing cell uptake, assaying gene knockdown, target site selection and more.
Interest in oligonucleotide synthesis has expanded dramatically since the publication of M. Gait's book in this series in 1984. The process has been automated, new reagents have been developed, and the range of uses for the compounds produced has increased and continues to grow. This volume provides practical guidance on oligonucleotide synthesis and methods for introducing modifications into these molecules. State- of-the-art techniques for automated synthesis are covered in two chapters on the production of oligodeoxynucleotides and oligoribonucleotides. Synthesis of modified oligodeoxynucleotides is also detailed, including modification of the phosphate backbone to produce phosphorothioates, phosphorodithiolates and methyl phosphonates, all of which are of considerable importance due to their potential therapeutic applications. Other chapters describe production of sugar-modified ilgodeoxynucleotides and the attachment of various reported groups; these techniques are useful to those interested in non-radioactive probes for hybridization and for the study of DNA-DNA and DNA protein interactions. This book will be of interest to academic and industrial researchers, enabling both chemists and non-chemists to synthesize oligonucleotides and analogues for a wide variety of experimental purposes.
Oligonucleotides represent one of the most significant pharmaceutical breakthroughs in recent years, showing great promise as diagnostic and therapeutic agents for malignant tumors, cardiovascular disease, diabetes, viral infections, and many other degenerative disorders. The Handbook of Analysis of Oligonucleotides and Related Products is an essential reference manual on the practical application of modern and emerging analytical techniques for the analysis of this unique class of compounds. A strong collaboration among thirty leading analytical scientists from around the world, the book provides readers with a comprehensive overview of the most commonly used analytical techniques and their advantages and limitations in assuring the identity, purity, quality, and strength of an oligonucleotide intended for therapeutic use. Topics discussed include: Strategies for enzymatic or chemical degradation of chemically modified oligonucleotides toward mass spectrometric sequencing Purity analysis by chromatographic or electrophoretic methods, including RP-HPLC, AX-HPLC, HILIC, SEC, and CGE Characterization of sequence-related impurities in oligonucleotides by mass spectrometry and chromatography Structure elucidation by spectroscopic methods (IR, NMR, MS) as well as base composition and thermal melt analysis (Tm) Approaches for the accurate determination of molar extinction coefficient of oligonucleotides Accurate determination of assay values Assessment of the overall quality of oligonucleotides, including microbial analysis and determination of residual solvents and heavy metals Strategies for determining the chemical stability of oligonucleotides The use of hybridization techniques for supporting pharmacokinetics and drug metabolism studies in preclinical and clinical development Guidance for the presentation of relevant analytical information towards meeting current regulatory expectations for oligonucleotide therapeutics This resource provides a practical guide for applying state-of-the-art analytical techniques in research, development, and manufacturing settings.