Engineered MRibonucleic Acid Regulation Using an Inducible Protein-Ribonucleic Acid Aptamer Interaction PDF Download
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Author: Brian Joshua Belmont
Publisher:
ISBN:
Category :
Languages : en
Pages : 131
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Book Description
The importance and pervasiveness of naturally occurring regulation of RNA function in biology is increasingly being recognized. A common regulatory mechanism uses inducible protein-RNA interactions to shape diverse aspects of cellular RNA fate. Recapitulating this using a novel set of protein-RNA interactions is appealing given the potential to subsequently modulate RNA biology in a manner decoupled from normal cellular physiology. We describe a ligand-responsive protein-RNA interaction module that can be used to target a specific RNA for subsequent regulation. Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, RNA aptamers binding to the bacterial Tet Repressor protein (TetR) with low- to sub- nanomolar affinities were identified. This interaction is reversibly controlled by tetracycline in a manner analogous to the interaction of TetR with its cognate DNA operator. Aptamer minimization and mutational analyses support a functional role for conserved sequence and structural motifs in TetR binding. We illustrate the utility of this chemically-inducible RNA-protein interaction to directly regulate translation in both a prokaryotic and eukaryotic organism. By genetically encoding TetR-binding RNA elements into the 5'-untranslated region (5'-UTR) of a given mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this modular system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from nonfunctional RNA-TetR interactions. We also demonstrate engineering this TetR-aptamer module to regulate subcellular mRNA localization. This is efficiently achieved by fusing TetR to proteins natively involved in localizing endogenous transcripts, and genetically encoding TetR-binding RNA aptamers into the target transcript. Using this platform, we achieve tetracycline-regulated enhancement of target transcript subcellular localization. We also systematically examine some rules for successfully forward engineering this RNA localization system. Altogether, these results define and validate an inducible protein-RNA interaction module that incorporates desirable aspects of a ubiquitous mechanism for regulating RNA function in Nature and that can be used as a foundation for functionally and reversibly controlling multiple fates of RNA in cells.
Author: Brian Joshua Belmont
Publisher:
ISBN:
Category :
Languages : en
Pages : 131
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Book Description
The importance and pervasiveness of naturally occurring regulation of RNA function in biology is increasingly being recognized. A common regulatory mechanism uses inducible protein-RNA interactions to shape diverse aspects of cellular RNA fate. Recapitulating this using a novel set of protein-RNA interactions is appealing given the potential to subsequently modulate RNA biology in a manner decoupled from normal cellular physiology. We describe a ligand-responsive protein-RNA interaction module that can be used to target a specific RNA for subsequent regulation. Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, RNA aptamers binding to the bacterial Tet Repressor protein (TetR) with low- to sub- nanomolar affinities were identified. This interaction is reversibly controlled by tetracycline in a manner analogous to the interaction of TetR with its cognate DNA operator. Aptamer minimization and mutational analyses support a functional role for conserved sequence and structural motifs in TetR binding. We illustrate the utility of this chemically-inducible RNA-protein interaction to directly regulate translation in both a prokaryotic and eukaryotic organism. By genetically encoding TetR-binding RNA elements into the 5'-untranslated region (5'-UTR) of a given mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this modular system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from nonfunctional RNA-TetR interactions. We also demonstrate engineering this TetR-aptamer module to regulate subcellular mRNA localization. This is efficiently achieved by fusing TetR to proteins natively involved in localizing endogenous transcripts, and genetically encoding TetR-binding RNA aptamers into the target transcript. Using this platform, we achieve tetracycline-regulated enhancement of target transcript subcellular localization. We also systematically examine some rules for successfully forward engineering this RNA localization system. Altogether, these results define and validate an inducible protein-RNA interaction module that incorporates desirable aspects of a ubiquitous mechanism for regulating RNA function in Nature and that can be used as a foundation for functionally and reversibly controlling multiple fates of RNA in cells.
Author: Robert E. Rhoads
Publisher: Humana
ISBN: 9781493936236
Category : Medical
Languages : en
Pages : 0
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Book Description
This volume presents detailed laboratory protocols for in vitro synthesis of mRNA with favorable properties, its introduction into cells by a variety of techniques, and the measurement of physiological and clinical consequences such as protein replacement and cancer immunotherapy. Synthetic techniques are described for structural features in mRNA that provide investigational tools such as fluorescence emission, click chemistry, photo-chemical crosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reduced activation of the innate immune response. Protocols are described for clinical applications such as large-scale transfection of dendritic cells, production of GMP-grade mRNA, redirecting T cell specificity, and use of molecular adjuvants for RNA vaccines. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resource for both laboratory investigators and clinicians interested in this powerful and rapidly evolving technology.
Author: Atsushi Ogawa
Publisher: Humana
ISBN: 9781493963300
Category : Science
Languages : en
Pages : 0
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Book Description
Artificial riboswitches and other ligand-responsive gene regulators make it possible to switch protein synthesis ON or OFF with arbitrary ligand molecules. Artificial Riboswitches: Methods and Protocols focuses on the state-of-the-art methods developed in recent years for creating artificial riboswitches, therefore this volume could be regarded as a collection of recipes for the gene circuit elements in synthetic biology and metabolic engineering. Chapters cover topics such as screening or rational design methods for obtaining artificial riboswitches that function in either bacterial or eukaryotic translational systems, protocols for evaluating the activities of the resultant riboswitches, as well as protocols for construction of ligand-dependent, trans-acting gene regulators. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Artificial Riboswitches: Methods and Protocols seeks to serve not only bioengineers who aim to reprogram cell behaviors and molecular biologists who leverage these regulators for genetic studies, but to all researchers interested in this fascinating field.
Author: Institute of Medicine
Publisher: National Academies Press
ISBN: 0309219396
Category : Science
Languages : en
Pages : 570
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Book Description
Many potential applications of synthetic and systems biology are relevant to the challenges associated with the detection, surveillance, and responses to emerging and re-emerging infectious diseases. On March 14 and 15, 2011, the Institute of Medicine's (IOM's) Forum on Microbial Threats convened a public workshop in Washington, DC, to explore the current state of the science of synthetic biology, including its dependency on systems biology; discussed the different approaches that scientists are taking to engineer, or reengineer, biological systems; and discussed how the tools and approaches of synthetic and systems biology were being applied to mitigate the risks associated with emerging infectious diseases. The Science and Applications of Synthetic and Systems Biology is organized into sections as a topic-by-topic distillation of the presentations and discussions that took place at the workshop. Its purpose is to present information from relevant experience, to delineate a range of pivotal issues and their respective challenges, and to offer differing perspectives on the topic as discussed and described by the workshop participants. This report also includes a collection of individually authored papers and commentary.
Author: Sven Klussmann
Publisher: John Wiley & Sons
ISBN: 3527607919
Category : Science
Languages : en
Pages : 518
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Book Description
In The Aptamer Handbook, leading scientists from academia as well as biotech and pharma companies introduce the revolutionary concept of designing RNA and DNA oligonucleotides with novel functions by in vitro selection. These functions comprise high affinity binding (aptamers), catalytic activity (ribozymes and deoxyribozymes) or combinations of binding and catalytic properties (aptazymes). Basic concepts and technologies describing in detail how these functional oligonucleotides can be identified are presented. Numerous examples demonstrate the versatility of in vitro selected oligonucleotides. Special emphasis has been put on a section that shows the broad applicability of aptamers, e. g. in target validation, for analytics, or as new therapeutics. This first overview in the field is of prime interest for a broad audience of scientists both in academia and in industry who wish to expand their knowledge on the potential of new oligonucleotide functions and their applications.
Author: Bin Wang
Publisher: CRC Press
ISBN: 9814411647
Category : Medical
Languages : en
Pages : 468
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Book Description
In the past few decades there has been incredible growth in "bionano"-related research, which has been accompanied by numerous publications in this field. Although various compilations address topics related to deoxyribonucleic acid (DNA) and protein, there are few books that focus on determining the structure of ribonucleic acid (RNA) and using RNA as building blocks to construct nanoarchitectures for biomedical and healthcare applications. RNA Nanotechnology is a comprehensive volume that details both the traditional approaches and the latest developments in the field of RNA-related technology. This book targets a wide audience: a broad introduction provides a solid academic background for students, researchers, and scientists who are unfamiliar with the subject, while the in-depth descriptions and discussions are useful for advanced professionals. The book opens with reviews on the basic aspects of RNA biology, computational approaches for predicting RNA structures, and traditional and emerging experimental approaches for probing RNA structures. This section is followed by explorations of the latest research and discoveries in RNA nanotechnology, including the design and construction of RNA-based nanostructures. The final segment of the book includes descriptions and discussions of the potential biological and therapeutic applications of small RNA molecules, such as small/short interfering RNAs (siRNAs), microRNAs (miRNAs), RNA aptamers, and ribozymes.
Author: Phoebe A. Rice
Publisher: Royal Society of Chemistry
ISBN: 0854042725
Category : Science
Languages : en
Pages : 417
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Book Description
This book provides both in-depth background and up-to-date information in this area. The chapters are organized by general themes and principles, written by experts who illustrate topics with current findings. Topics covered include: - the role of ions and hydration in protein-nucleic acid interactions - transcription factors and combinatorial specificity - indirect readout of DNA sequence - single-stranded nucleic acid binding proteins - nucleic acid junctions and proteins, - RNA protein recognition - recognition of DNA damage. It will be a key reference for both advanced students and established scientists wishing to broaden their horizons.
Author: Ron Milo
Publisher: Garland Science
ISBN: 1317230698
Category : Science
Languages : en
Pages : 400
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Book Description
A Top 25 CHOICE 2016 Title, and recipient of the CHOICE Outstanding Academic Title (OAT) Award. How much energy is released in ATP hydrolysis? How many mRNAs are in a cell? How genetically similar are two random people? What is faster, transcription or translation?Cell Biology by the Numbers explores these questions and dozens of others provid
Author:
Publisher: Academic Press
ISBN: 0128171944
Category : Medical
Languages : en
Pages : 444
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Book Description
RNA-based Regulation in Human Health and Disease offers an in-depth exploration of RNA mediated genome regulation at different hierarchies. Beginning with multitude of canonical and non-canonical RNA populations, especially noncoding RNA in human physiology and evolution, further sections examine the various classes of RNAs (from small to large noncoding and extracellular RNAs), functional categories of RNA regulation (RNA-binding proteins, alternative splicing, RNA editing, antisense transcripts and RNA G-quadruplexes), dynamic aspects of RNA regulation modulating physiological homeostasis (aging), role of RNA beyond humans, tools and technologies for RNA research (wet lab and computational) and future prospects for RNA-based diagnostics and therapeutics. One of the core strengths of the book includes spectrum of disease-specific chapters from experts in the field highlighting RNA-based regulation in metabolic & neurodegenerative disorders, cancer, inflammatory disease, viral and bacterial infections. We hope the book helps researchers, students and clinicians appreciate the role of RNA-based regulation in genome regulation, aiding the development of useful biomarkers for prognosis, diagnosis, and novel RNA-based therapeutics. Comprehensive information of non-canonical RNA-based genome regulation modulating human health and disease Defines RNA classes with special emphasis on unexplored world of noncoding RNA at different hierarchies Disease specific role of RNA - causal, prognostic, diagnostic and therapeutic Features contributions from leading experts in the field
Author: Kiyoshi Nagai
Publisher: Oxford University Press, USA
ISBN:
Category : Medical
Languages : en
Pages : 302
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Book Description
The study of RNA-protein interactions is crucial to understanding the mechanisms and control of gene expression and protein synthesis. The realization that RNAs are often far more biologically active than was previously appreciated has stimulated a great deal of new research in this field. Uniquely, in this book, the world's leading researchers have collaborated to produce a comprehensive and current review of RNA-protein interactions for all scientists working in this area. Timely, comprehensive, and authoritative, this new Frontiers title will be invaluable for all researchers in molecular biology, biochemistry and structural biology.
