Download or read book Design and Evolution of Trans Splicing Group I Intron Ribozymes written by Zhaleh N. Amini and published by . This book was released on 2015 with total page 151 pages. Available in PDF, EPUB and Kindle. Book excerpt: Group I introns are catalytic RNAs (ribozymes) capable of catalyzing their self-excision from precursor RNAs through two consecutive transesterification reactions. Although the ribozyme has evolved to perform this cis-splicing reaction in nature, man-made modifications to the 5' end of the ribozyme have allowed it to catalyze a trans-splicing reaction, in which it is able to replace the 3' portion of a substrate RNA with it's own 3' tail. The trans-splicing group I introns used in this thesis were variants of the Tth.L 1925 IC1 group I intron ribozyme found in Tetrahymena thermophila. The work contained in this dissertation aims to both utilize trans-splicing group I introns to further understand principles of RNA evolution, as well as develop and optimize a new trans-splicing variant of the ribozyme for future use in therapy. In the first study of this dissertation, a trans-splicing group I intron was used as a model system to examine the effect of selection pressure and recombination on evolving populations of RNA in a cellular environment. Four parallel evolutions were completed, two employing a low selection pressure, and two employing a high selection pressure. Ribozyme populations with higher efficiency, measured by cellular growth conferred by the ribozyme, resulted from evolutions performed at a low selection pressure. It was found that this increase in fitness was the result of a set of four mutations acting cooperatively. Fitness profiles of evolutionary intermediates revealed that a low selection pressure can increase the accessibility of evolutionary paths leading to the evolution of cooperative mutations. This finding not only adds to the understanding of natural RNA evolution, but also aids in the design of more efficient evolutions of RNA species. In the second study of this dissertation, a new trans-splicing variant of the group I intron was developed, capable of catalyzing the removal of internal sequences from pre-mRNA and joining the two flanking sequences, thereby generating a functional RNA. This group I intron has been termed the `spliceozyme' because its action is analogous to that of the spliceosome. The action performed by the spliceozyme give this system the ability to repair certain types of diseases caused by mis-splicing and therefore, the potential to be used therapeutically. To increase the efficiency and therapeutic potential of this system, the spliceozyme was evolved in E. coli cells, challenging it to more efficiently catalyze the removal of internal sequences. The most efficient variant contained a set of mutations resulting in increased product formation and decreased side product formation. This observed effect was seen in vitro, suggesting that this effect may increase spliceozyme efficiency in a range cell types. Future work will move this system into mammalian cells and optimize the spliceozyme for use in a mammalian system, thus developing it as a therapeutic tool.
Download or read book In Vivo Evolution of a Trans Splicing Group I Intron Ribozyme written by Ishani Behera and published by . This book was released on 2018 with total page 49 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Tetrahymena group I intron was one of the two first catalytic RNAs (ribozymes) to be discovered. Group I introns are sequences that can exist between exons in pre-mRNAs, that are able to self-splice and remove themselves when forming mature mRNAs in the absence of the spliceosome. The Tetrahymena cis-splicing ribozyme was engineered to accept substrate RNAs in trans. The designed trans-splicing ribozyme was termed the "spliceozyme". The spliceozyme could in principle be used in therapeutic applications. However, there are two hurdles to overcome; the delivery of the ribozyme into cells and its efficiency once delivered. The focus of this study is to improve the efficiency of the trans-splicing ribozyme in cells by evolving the ribozyme in E. coli. In a previous study, an evolution system in cells was developed for the spliceozyme which was used to generate a clone, W11, that increased product formation. However, this evolution used high ribozyme expression levels and focused on a single splice site. To improve the efficiency and sequence generality, the spliceozyme was evolved at low expression levels at two different splice sites with different flanking sequences. The results of the spliceozyme evolution in bacterial cells showed that specific mutations are able to improve spliceozyme efficiency in bacterial cells. This suggests that further analysis, and perhaps further evolution experiments, could generate spliceozymes with improved efficiency on a broad range of substrate sequences.
Download or read book In Vivo Selection and in Vivo Evolution of a Trans splicing Ribozyme written by Karen Elizabeth Olson and published by . This book was released on 2012 with total page 199 pages. Available in PDF, EPUB and Kindle. Book excerpt: Group I introns are catalytic RNAs (ribozymes) that are capable of self-splicing out of primary transcripts. Through two consecutive transesterification reactions, the intron is removed and flanking exons are ligated. The group I intron sequence evolved for cis-splicing in nature. However, sequence modifications allow this ribozyme to catalyze a trans-splicing reaction as well. If the 5'-portion of the intron is truncated so that it no longer contains an exon, the ribozyme can then bind in trans at its 5'-end to an RNA substrate, cleave the substrate and replace the 3'-terminal portion of the substrate with its own 3'-exon. When the splice site is upstream of a mutation in a messenger RNA (mRNA) sequence, the transfer of the 3'-exon can replace and repair the downstream portion of the mRNA. Thus, this reaction can be utilized as a type of gene therapy on the mRNA level. Repairing mRNA offers the benefit of maintaining natural gene regulation and the ability to target gain-of-function mutations that gene therapy via gene transfer does not. Although the idea of utilizing a group I intron for gene therapy is not new, to date no gene therapy trials have used this trans-splicing reaction. This is in part due to the low efficiency of the ribozyme under clinically relevant conditions. This thesis represents an effort to increase trans-splicing efficiency of the Tetrahymena thermophila group I intron toward the development of a gene therapy. To find ribozyme variants with increased trans-splicing efficiency an in vivo selection procedure was developed in which the most efficient ribozymes are selected from millions of ribozyme variants. Ribozymes with an improved 5'-terminus were selected from a library with 9 x 106 ribozyme variants. Ribozymes with an improved internal sequence resulted from 21 rounds of evolution, which included mutagenesis and recombination. The in vivo evolution produced a highly efficient repair ribozyme that appears to be recruiting a cellular protein to increase activity. Future experiments that establish an analogous selection strategy in mammalian cells may make it possible to develop trans-splicing ribozymes for treating genetic diseases.
Download or read book Ribozymes and RNA Catalysis written by David Malcolm James Lilley and published by Royal Society of Chemistry. This book was released on 2008 with total page 339 pages. Available in PDF, EPUB and Kindle. Book excerpt: Takes the reader through the origins of catalysis in RNA and necessarily includes significant discussion of structure and folding. The main focus of the book concerns chemical mechanism with extensive comment on how, despite the importance of RNA catalysis in the cell, its origins are still poorly understood and often controversial. The reader is given an outline of the important role of RNA catalysis in many aspects of cell function, including RNA processing and translation.
Download or read book Better Ribozymes for Trans splicing and Triphosphorylation written by Gregory F. Dolan and published by . This book was released on 2015 with total page 128 pages. Available in PDF, EPUB and Kindle. Book excerpt: Catalytic RNAs (ribozymes) are RNAs that can catalyze a chemical reaction without the use of protein cofactors. This dissertation centered on improving the activities for two different ribozymes: a trans-splicing group I intron ribozyme and a triphosphorylation ribozyme. Trans-splicing group I intron ribozymes can be used in gene therapy and synthetic biology applications, and this work focused on improving the trans-splicing activity of the group I intron ribozyme from Azoarcus, a small and robustly folding ribozyme located in the intervening sequence of pre-tRNAIle. These results showed that although the trans-splicing Azoarcus ribozyme could not mediate a growth phenotype in E. coli cells, the Azoarcus ribozyme is indeed capable of catalyzing the in vitro trans-splicing reaction as efficiently as other trans-splicing ribozymes previously tested. The most efficient trans-splicing Azoarcus ribozymes contained secondary structural contacts that mimicked the contacts made by the natural cis-splicing ribozyme, suggesting that the natural splicing interactions are the preferred trans-splicing interactions for the Azoarcus ribozyme. The second ribozyme improved in this dissertation is the triphosphorylation ribozyme, a ribozyme that triphosphorylates the 5'-hydroxyl of RNAs using trimetaphosphate as a substrate. This ribozyme's activity is important in the context of the RNA world and in the origin of life because RNA polymerization is an energetically unfavorable process in an aqueous environment, and the 5'-triphsophate can supply the energetic driving force for RNA polymerization. A previous graduate student in the Müller laboratory performed an in vitro selection identifying the first triphosphorylation ribozymes. The work herein improved the activity of one of the most active original triphosphorylation ribozymes by performing a 'doped selection.' A `doped selection' is a method to identify sequence variants with higher activity by selecting for the best ribozymes from a partially randomized pool consisting of sequence variants of the original ribozyme. This selection yielded a ribozyme with seven mutations and a catalytic rate that was 24-fold higher then the first generation ribozyme. Unlike the original ribozyme, this new ribozyme has measurable activity at prebiotically relevant trimetaphosphate concentrations and has a catalytic rate of 6.8 min−1 at optimal conditions.
Download or read book Tripartite Trans splicing of the L1 LtrB Group Ll Intron written by Christine Elizabeth Ritlop and published by . This book was released on 2009 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Handbook of RNA Biochemistry written by Roland K. Hartmann and published by John Wiley & Sons. This book was released on 2015-06-22 with total page 1370 pages. Available in PDF, EPUB and Kindle. Book excerpt: The second edition of a highly acclaimed handbook and ready reference. Unmatched in its breadth and quality, around 100 specialists from all over the world share their up-to-date expertise and experiences, including hundreds of protocols, complete with explanations, and hitherto unpublished troubleshooting hints. They cover all modern techniques for the handling, analysis and modification of RNAs and their complexes with proteins. Throughout, they bear the practising bench scientist in mind, providing quick and reliable access to a plethora of solutions for practical questions of RNA research, ranging from simple to highly complex. This broad scope allows the treatment of specialized methods side by side with basic biochemical techniques, making the book a real treasure trove for every researcher experimenting with RNA.
Download or read book Progress in Molecular Biology and Translational Science written by David B. Teplow and published by Academic Press. This book was released on 2018-10-16 with total page 218 pages. Available in PDF, EPUB and Kindle. Book excerpt: Progress in Molecular Biology and Translational Science, Volume 159, provides the most topical, informative and exciting monographs available on a wide variety of research topics related to prions, viruses, bacteria and eukaryotes. The series includes in-depth knowledge on molecular biological aspects of organismal physiology, along with insights on how this knowledge may be applied to understand and ameliorate human disease. New chapters in this release discuss timely topics, such as Targeting recently deorphanized GPR83 for the treatment of infection, stress, and drug addiction, Arrestin Structure-Function, Arrestins in the Cardiovascular System, Analysis of biased agonism, and more. Includes comprehensive coverage of molecular biology Presents ample use of tables, diagrams, schemata, and color figures to enhance the reader's ability to rapidly grasp the information provided Contains contributions from renowned experts in the field
Download or read book Ribozymes and siRNA protocols written by Mouldy Sioud and published by Humana Press. This book was released on 2004-03-05 with total page 624 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this completely updated and expanded edition of a classic bench manual, hands-on experts take advantage of the latest advances in ribozyme, DNAzyme, 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 hairpain 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 ribozymes protocols for effective design, selection, and therapeutic applications; and the latest RNAi methods for sequencing-specific gene silencing in a wide variety of organisms. Comprehensive and up-to-date, Ribozymes and siRNA Protocols synthesizes for experienced and novice investigators alike the exciting advances in understanding nucleic acid enzymes and demonstrates how they may be used to analyze gene function and target validation, and to productively develop new therapeutics for human diseases.
Download or read book Chemistry of Nucleic Acids written by Harri Lönnberg and published by Walter de Gruyter GmbH & Co KG. This book was released on 2024-09-23 with total page 553 pages. Available in PDF, EPUB and Kindle. Book excerpt: Life in all its forms is based on nucleic acids which store and transfer genetic information. The book addresses main aspects of synthesis, hydrolytic stability and solution equilibria of nucleosides, nucleotides and oligonucleotides, as well as synthesis of their structural analogs that are of interest in chemotherapy. In addition, recent achievements in chemistry of catalytic nucleic acids, development of oligonucleotide based drugs and novel strategies for their targeting and delivery are discussed. The central theme always is the correlation of structure and function.
Download or read book Structure and Conformational Rearrangements During Splicing of the Ribozyme Component of Group II Introns written by Cheng-Fang Li and published by . This book was released on 2011 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Group II introns are a class of RNAs best known for their ribozyme-catalyzed, self-splicing reaction. Under certain conditions, the introns can excise themselves from precursor mRNAs and ligate together their flanking exons, without the aid of proteins. Group II introns generally excise from pre-mRNA as a lariat, like the one formed by spliceosomal introns, similarities in the splicing mechanism suggest that group II introns and nuclear spliceosomal introns may share a common evolutionary ancestor.Despite their very diverse primary sequences, group II introns are defined by a highly conserved secondary structure. This generally consists of six domains (Domain I-Domain VI; D1-D6) radiating from a central wheel. Each of the six intronic domains has a specific role in folding, conformational rearrangements or catalysis. The native conformation of a group II intron is sustained by intra- and interdomain long-range tertiary interactions, which are critical either for folding of the intron to the native state or for its catalytic activity. In brief, Domain V interacts with Domain I to form the minimal catalytic core; Domain VI contains a highly conserved bulged adenosine serving as the branch-point nucleotide. DII and Domain III contribute to RNA folding and catalytic efficiency. Domain IV, which encodes the intron ORF, is dispensable for ribozyme activity.Group II intron splicing proceeds through two step transesterification reactions which yield ligated exons and an excised intron lariat. It is initiated by the 2'-hydroxyl group of the bulged adenosine within Domain 6, which serves as a branch point and attacks the phosphate at the 5'-end of the intron, thus releasing the 5'-exon while forming a lariat structure in the first step. The released 5'-exon, which is bound to the intron through base pairing interactions, is then positioned correctly to attack the 3'-splice site with its free 3'-OH in the second step of splicing. It is generally believed that the structure of a group II ribozyme undergoes conformational rearrangements between first step and second step and domain VI must play a central role in the process. However, despite the identification of several interdomain tertiary interactions, neither NMR nor chemical probing studies have been successful in determining the local surroundings of the branch-point adenosine and neighboring domain VI nucleotides in the ribozyme active site. By using phylogenetic analysis and molecular modelling, we have identified several areas of the molecule which have the potential to constitute the docking site of domain VI. Mutations were introduced in putative binding sites and the resulting, mutant RNAs have been kinetically characterized. This has allowed us to identify a site within the ribozyme that appears to be specifically involved in the branching reaction. In order to further investigate the interaction between that site and domain VI, we set up a system in which the docking of domain VI into its presumed binding site is ensured by the addition of DNA/RNA oligos that position the two RNA elements in an appropriate orientation. By combining the information from such experiments, we have built an atomic-resolution model of the complex formed by domain VI, the branch site and the rest of the intron at the time at which splicing is initiated.
Download or read book Biochemistry and Molecular Biology Compendium written by Roger L. Lundblad and published by CRC Press. This book was released on 2019-11-11 with total page 551 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book is an accessible resource offering practical information not found in more database-oriented resources. The first chapter lists acronyms with definitions, and a glossary of terms and subjects used in biochemistry, molecular biology, biotechnology, proteomics, genomics, and systems biology. There follows chapters on chemicals employed in biochemistry and molecular biology, complete with properties and structure drawings. Researchers will find this book to be a valuable tool that will save them time, as well as provide essential links to the roots of their science. Key selling features: Contains an extensive list of commonly used acronyms with definitions Offers a highly readable glossary for systems and techniques Provides comprehensive information for the validation of biotechnology assays and manufacturing processes Includes a list of Log P values, water solubility, and molecular weight for selected chemicals Gives a detailed listing of protease inhibitors and cocktails, as well as a list of buffers
Download or read book Therapeutic Applications of Ribozymes written by Kevin J. Scanlon and published by Springer Science & Business Media. This book was released on 1998-06-23 with total page 488 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Download or read book Ribozymes written by Sabine Müller and published by John Wiley & Sons. This book was released on 2021-07-09 with total page 81 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ribozymes Provides comprehensive coverage of a core field in the molecular biosciences, bringing together decades of knowledge from the world’s top professionals in the field Timely and unique in its breadth of content, this all-encompassing and authoritative reference on ribozymes documents the great diversity of nucleic acid-based catalysis. It integrates the knowledge gained over the past 35 years in the field and features contributions from virtually every leading expert on the subject. Ribozymes is organized into six major parts. It starts by describing general principles and strategies of nucleic acid catalysis. It then introduces naturally occurring ribozymes and includes the search for new catalytic motifs or novel genomic locations of known motifs. Next, it covers the development and design of engineered ribozymes, before moving on to DNAzymes as a close relative of ribozymes. The next part examines the use of ribozymes for medicinal and environmental diagnostics, as well as for therapeutic tools. It finishes with a look at the tools and methods in ribozyme research, including the techniques and assays for structural and functional characterization of nucleic acid catalysts. The first reference to tie together all aspects of the multi-faceted field of ribozymes Features more than 30 comprehensive chapters in two volumes Covers the chemical principles of RNA catalysis; naturally occurring ribozymes, engineered ribozymes; DNAzymes; ribozymes as tools in diagnostics and therapy, and tools and methods to study ribozymes Includes first-hand accounts of concepts, techniques, and applications by a team of top international experts from leading academic institutions Dedicates half of its content to methods and practical applications, ranging from bioanalytical tools to medical diagnostics to therapeutics Ribozymes is an unmatched resource for all biochemists, biotechnologists, molecular biologists, and bioengineers interested in the topic.
Download or read book Applied RNA Bioscience written by Seiji Masuda and published by Springer. This book was released on 2018-04-10 with total page 286 pages. Available in PDF, EPUB and Kindle. Book excerpt: The focus of this book is to introduce up-to-date information on applications and practical use of RNA for agriculture, biotechnology and medicine. It provides unique ideas, tools, and methods in detail from a variety of scientific and technical disciplines. RNA science has progressed enormously in recent decades, and vast amounts of information on RNA functions and their regulatory mechanisms are becoming available. Such a progress opened the door to an age of practical application of RNA in many fields including agriculture, plant science, medical science, brewing and fermentation technology, and material production. This book inspires its readership and contributes to not only expansion in application of RNA but also to basic research.
Download or read book Synthetic Biology written by Christina Smolke and published by John Wiley & Sons. This book was released on 2018-02-28 with total page 532 pages. Available in PDF, EPUB and Kindle. Book excerpt: A review of the interdisciplinary field of synthetic biology, from genome design to spatial engineering. Written by an international panel of experts, Synthetic Biology draws from various areas of research in biology and engineering and explores the current applications to provide an authoritative overview of this burgeoning field. The text reviews the synthesis of DNA and genome engineering and offers a discussion of the parts and devices that control protein expression and activity. The authors include information on the devices that support spatial engineering, RNA switches and explore the early applications of synthetic biology in protein synthesis, generation of pathway libraries, and immunotherapy. Filled with the most recent research, compelling discussions, and unique perspectives, Synthetic Biology offers an important resource for understanding how this new branch of science can improve on applications for industry or biological research.
Download or read book Transcription and Splicing written by B. D. Hames and published by Oxford University Press, USA. This book was released on 1988 with total page 238 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book gives a co-ordinated review of our present knowledge of eukaryotic RNA synthesis.
