Antisense and ribozymes have a relatively short yet successful history as research tools in gene expression studies, and thus are considered as having high potential reagents in treating viral infections and cancer. This laboratory companion provides detailed information on the potential, advantages and limitations of this methodology. It critically discusses potential pitfalls, presents strategies for choosing targets and delivery systems, so as to allow the selection of the optimum methodology for achieving fast and reliable experimental success with any human or other biological system. For researchers, technicians and advanced graduates in experimental medicine, molecular and cell biology.

In this completely updated and expanded edition of a classic bench manual, hands-on experts take advantage of the latest advances in ribozyme, DNAzyme, hammerhead ribozymes and derivatives, and RNA interference technologies to describe in detail the exciting and successful methods now available for gene inactivation in vitro and in vivo. Their optimized techniques employ hairpin ribozymes, DNAzymes, hammerhead ribozymes and derivatives, group I intron ribozymes, RNase P ribozymes, and siRNAs, as well as general methods for RNA structure analysis, delivery of oligonucleotides, and gene therapy. Also provided are novel methods for identifying accessible cellular mRNA sites; group I intron and RNAse P ribozyme protocols for effective design, selection, and therapeutic applications; and the latest RNAi methods for sequence-specific gene silencing in a wide variety of organisms. Additional techniques cover the analysis of ribozyme structures and conformational transitions using nucleotide analog interference mapping and fluorescence resonance energy transfer, the use of ribozymes in clinical and gene therapy, and the use of ribozymes and DNAzymes in rodent models of human disease. Each proven protocol includes a background introduction outlining the principle behind the technique, step-by-step instructions, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and up-to-date, Ribozymes and siRNA Protocols details for experienced and novice investigators alike the many exciting advances in our understanding of nucleic acid enzymes, as well as demonstrating how they may be used to analyze gene function and target validation, and to productively develop novel therapeutics for human diseases.

Antisense technology is a powerful procedure that permits the controlled silencing of a specific gene for investigations of mRNA and protein function. This valuable text provides proven step-by-step protocols for antisense techniques in a range of different organisms and cell culture systems. In addition it discusses the potential benefits and problems for various antisense methods which complement gene knock-out experiments. The book includes antisense techniques such as: analysis of nucleic acid structures; measurement and evaluation of antisense effects; selection, preparation and the use of antisense oligonucleotides; in vitro RNA transcription; construction strategies, testing and optimization of catalytic antisense RNAs based on hammerhead ribozymes; synthesis and evaluation of 2-5 A- antisense chimeras for targeted degradation of RNA. The application of these technologies are then described, with chapters on antisense techniques in IDictyostelium, plants and the medical uses and benefits of antisense sense technology both in vitro and in vivo. Finally, non-antisense effects of oligonucleotides, "anti-sense rescue", shotgun antisense mutagenesis, and in vivo, in vitro and in situ detection of sense: antisense duplexes by structure-specific anti-RNA antibodies are discussed. The numerous hints and tips for success and advice on trouble shooting, make this authoritative text invaluable to all those researchers who work on gene expression, RNA transcription, or protein function.

RNA technologies are the driving forces of modern medicine and biotechnology. They combine the fields of biochemistry, chemistry, molecular biology, cell biology, physics, nanotechnology and bioinformatics. The combination of these topics is set to revolutionize the medicine of tomorrow. After more than 15 years of extensive research in the field of RNA technologies, the first therapeutics are ready to reach the first patients. Thus we are witnessing the birth of a very exciting time in the development of molecular medicine, which will be based on the methods of RNA technologies. This volume is the first of a series. It covers various aspects of RNA interference and microRNAs, although antisense RNA applications, hammerhead ribozyme structure and function as well as non-coding RNAs are also discussed. The authors are internationally highly respected experts in the field of RNA technologies.

In recent years, unprecedented advances in many aspects of the molecular biology of nucleic acids have been witnessed. The area of RNA chemistry has undergone a kind of explosion, with a huge interest in RNA-mediated catalysis. At the same time, our structural understanding of DNA-protein interactions has increased enormously, and the related area of RNA-protein interactions is beginning to gather pace. This softcover edition from the successful series Nucleic Acids and Molecular Biology is devoted to the structure and mechanism of ribozymes, and their potential exploitation. The subject has both important evolutionary implications and potential practical application in the development of therapeutic agents for diseases such as AIDS.

Examines the biology of antisense oligodeoxynucleotides, RNA and ribozymes, and explores their potential therapeutic applications, particularly for inhibition of viral replication and cancer gene expression. Close attention is also given to drug approval.

In Therapeutic Applications of Ribozymes, expert laboratory scientists describe in detail their methodologies for constructing ribozymes designed to elucidate the role of specific genes as key routes to the development of novel therapies for a wide variety of diseases. The authors review the many sites targeted with ribozymes in various diseases and provide specific accounts of the practical techniques required for the proper use of ribozymes in these systems. Their cutting-edge protocols demonstrate how to achieve ribozyme expression in distinct cellular systems, the preparation and use of high-efficiency ribozyme DNA or RNA delivery, and the studies required to prove the efficacy of ribozyme-mediated inhibition, with its concomitant effect on phenotypic parameters. Therapeutic Applications of Ribozymes contains all the practical detail necessary to realize the high promise of ribozyme technology as a significant, widely used methodology. Focused on ribozyme targets and the novel associated technology, the book immediately becomes the leading resource for all those seeking to transform this new category of therapeutic agent into efficacious clinical practice.

Advances in Nanomedicine for the Delivery of Therapeutic Nucleic Acids addresses several issues related to safe and effective delivery of nucleic acids (NAs) using nanoparticles. A further emphasis would be laid on the mechanism of delivery of NAs, the barriers encountered and the strategies adapted to combat them. An exhaustive account of the advantages as well shortcomings of all the delivery vectors being employed in delivery of various NAs will be provided. On final note the regulatory aspects of nanoparticles mediated NA would be discussed, with focus on their clinical relevance. The design and development of nucleic acid-based therapeutics for the treatment of diseases arising from genetic abnormalities has made significant progress over the past few years. NAs have been widely explored for the treatment of cancer and infectious diseases or to block cell proliferation and thereby caused diseases. Advances in synthetic oligonucleotide chemistry resulted in synthesis of NAs that are relatively stable in in vivo environments. However, cellular targeting and intracellular delivery of NAs still remains a challenge. Further development of NA-based therapeutics depends on the progress of safe and effective carriers for systemic administration. Nanomedicine has facilitated availability of vectors with diminished cytotoxicity and enhanced efficacy which are rapidly emerging as systems of choice. These vectors protect NAs from enzymatic degradation by forming condensed complexes along with targeted tissue and cellular delivery. During the past few years, a myriad reports have appeared reporting delivery of NAs mediated by nanoparticles. This book will provide an overview of nanoparticles being employed in the in vitro and in vivo delivery of therapeutically relevant NAs like DNA, siRNA, LNA, PNA, etc. - Provides a complete overview of the applicatiosn of nanomedicine in the delivery of nucleic acids, from characterization of nanoparticles, to in vitro and in vivo studies - Discusses delivery issues of less well explored nucleic acids, like PNAs, Ribozymes, DNAzymes, etc. - Summarizes the current state of research in nucleic acid delivery and underscores the future of nanomedicine in this field