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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.
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.
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.
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 thier potential therapeutic applications. Other chapters decsribe 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.
This book compiles recent research on the modification of nucleic acids. It covers backbone modifications and conjugation of lipids, peptides and proteins to oligonucleotides and their therapeutic use. Synthesis and application in biomedicine and nanotechnology of aptamers, fluorescent and xeno nucleic acids, DNA repair and artificial DNA are discussed as well.
Provides practical guidance on the synthesis and chemical modification of oligonucleotides. These molecules have a wide variety of experimental uses, including use as potential therapeutics and in non-radioactive probing for the study of DNA-DNA and DNA-protein interactions.
The use of oligonucleotides as therapeutic agents rests upon their ability to interfere, in a sequence-specific manner, with the fundamental machinery of protein synthesis either by binding to the mRNAs transcribed from a gene or by binding directly to a target gene. This approach can be used not only for inhibition of the synthesis of host proteins but also of those required by invading pathogens. Potential therapeutic applications are enormous, ranging over hypertension, cardiovascular disease, autoimmune disease, vital and other parasitic infections (especially HIV), and cancer. This book discusses the chemistry and pharmacokinetics of oligonucleotides and their analogues, and surveys the results of structure-activity studies and current clinical trials. It also critically reviews the problems with antisense therapy, such as the enzymatic destruction of oligonucleotides, the doses required for a therapeutic response, the difficulty in directing oligonucleotides to particular target tissues and cells, the need for parenteral administration, and doubts concerning the mechanism of action (especially problems associated with non-specific binding to proteins) and long-term effects.
This book provides a compelling overall update on current status of RNA interference
"Synthetic short double-stranded small interfering RNAs (siRNAs) have the capacity to inhibit gene expression of a gene target; unfortunately, these RNAs display unfavorable metabolic stability and pharmacokinetic properties. In order to overcome these hurdles, it is possible to chemically alter the nucleotides that comprise the siRNA therapeutic to confer increased nuclease stability as well as alter their binding properties. It is also possible to use delivery vehicles, such as nanoparticles, to help improve their delivery into target cells. Therefore, in chapter 2 the creation of novel nucleoside analogues to first tackle the issue of metabolic stability will be reported; followed by chapter 3 which described the design of oligonucleotide-modified nanoparticles to increase cell permeation and allow for targeted delivery to DRR-expressing Glioblastoma multiformes cells. As such, we report on the synthesis of 2'-F,4'-OMe and 2',4'-diOMe ribo-uridine derivatives, their incorporation into siRNA duplexes, and the physicochemical and gene silencing properties of these novel 2',4'-modified siRNAs. Both nucleosides were prepared stereoselectively in 6 steps starting from 2'-F rU and 2'-OMe rU for 2'-F,4'-OMe and 2',4'-diOMe, respectively, and subsequently converted to the corresponding phosphoramidites through conventional oligonucleotide chemistry for incorporation into oligonucleotides through standard solid-phase chemistry. NMR analysis of these nucleosides revealed that the 4' substituent imparts a bias towards the North sugar pucker conformation in both cases. Incorporation of 2'-F,4'-OMe rU into DNA strands resulted in destabilizing thermal effects in DNA:DNA hybrids and slightly destabilizing effects in DNA:RNA hybrids, but neutral effects within modified RNA strands in RNA:RNA duplexes. Furthermore, the 2'-F,4'-OMe rU modification was shown to be tolerated in both the antisense and sense strand in siRNA-targeted luciferase gene knockdown, whereas the 2',4'-diOMe rU modification was shown to be tolerated in the sense strand only. The CD analysis of these same strands also confirmed that the incorporation of these novel modifications does not affect the formation of duplex; where the A-form duplex (typical of RNA duplexes) is observed. As for the nanoparticle design, a block co-polymer is functionalized with azide and furan groups on the corona to allow for conjugation of antisense oligonucleotides and antibodies targeting upregulated receptors in glioblastoma cells. In collaboration, we were able to create a modified vehicle strand necessary for conjugation to the nanoparticle corona, upon which we were successful in hybridizing the complementary antisense oligonucleotide. This duplex was then successfully conjugated to the azide groups on the surface of the nanoparticle by copper-free Click chemistry. Single-stranded and double-stranded (hybridized) antisense oligonucleotides were transfected into live DsredDRR cells and 50% knockdown of the DRR gene was observed. " --