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Author: Hua Shi
Publisher:
ISBN:
Category :
Languages : en
Pages : 324
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Book Description
Author: Hua Shi
Publisher:
ISBN:
Category :
Languages : en
Pages : 324
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Book Description
Author: Xiaochun Fan
Publisher:
ISBN:
Category :
Languages : en
Pages : 400
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Author: Lina Rajili Bagepalli
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
DNA binding transcription factors are integral to transcription activation and repression and in many cases are comprised of multiple domains and subunits. To elucidate the contributions of these subunits to the interactions that these factors make, surface specific inhibitors must be developed and characterized which have the potential to block a specific domain of the factor thereby impairing its activity. The Heat Shock Response is a highly conserved protective mechanism that is regulated at the transcriptional level by transcription factors called Heat Shock Factors (HSFs). When activated by high temperatures or proteotoxic stress, HSFs strongly induce the expression of Heat Shock genes (HS genes), that encode Heat Shock Proteins (HSPs). Of the family of HSFs in mammals, HSF1 is the functional homolog of the single HSF in yeast and fruit fly. HSF1 consists of at least five major domains; the DNA Binding Domain (DBD), Trimerization Domain (TD), the Regulatory Domain (RD), the Leucine Zipper Regulatory Domain (HR-C) and the Trans-Activation Domain (TAD) which function to coordinate HSF1's ability to bind to its target DNA elements and trans-activate HS genes under heat shock conditions. To elucidate the mechanistic roles of each domain in living cells, I have used the novel approach of blocking domains with RNA aptamers. RNA aptamers are short, single stranded RNAs that bind specifically and with high affinity to their selected target, whether it is a protein domain, transcription factor or small molecule. We have thousands of selected candidate aptamers for HSF1, however, we needed to identify those that have domain specific binding in order to test a domain specific effect. Initially, to identify the binding affinities of the selected RNA Aptamers, I used Electrophoretic Mobility Shift Assays (EMSA). As part of this study, I have screened aptamers that are able to bind hHSF1 strongly and identified the binding regions of some high-affinity candidates. Many of the aptamers directly interact with a minimal hHSF1 containing the regulatory, trimerization and DNA-binding domains. Of the high-affinity binders only the top-aptamer partially competes with hHSF1 for binding to a heat shock element. To identify a genome-wide effect of the characterized top aptamer which has some competitive ability, I performed a PRO-seq experiment. We tried this experiment multiple times but were unable to measure a genome-wide effect. To improve the ability of these RNA aptamers as molecular inhibitors, I used RNA scaffolds to generate multimeric aptamers using the minimal core aptamers. Dimerization and trimerization enhances the competitive behavior of the monomeric aptamers up to 70-fold. Overall, we have generated and optimized high-affinity tools to interfere with the DNA-binding activity of hHSF1 that provide further opportunities for precisely disrupting the functions of hHSF1 in a biological context.
Author: Chris Baugh
Publisher:
ISBN:
Category : Protein binding
Languages : en
Pages : 348
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Author: Brian Joshua Belmont
Publisher:
ISBN:
Category :
Languages : en
Pages : 131
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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: Andreas Werner
Publisher:
ISBN:
Category :
Languages : en
Pages : 211
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Book Description
The research in this Ph.D. thesis is part of an ongoing effort to improve our understanding of RNA structure and stability: In the first part, an RNA aptamer is studied in complex with its target, the Sec7 domain of intracellular protein cytohesin-1. Aptamers are in vitro selected RNA molecules that have an affinity rivaling that of monoclonal antibodies, possess interesting pharmacological properties and are a powerful tool for functional analysis in vivo. We also present a systematic structural investigation of how aptamers and their natural counterparts bind their ligands in order to delineate common principles. In the second part, we study the counterion-induced collapse of prokaryotic RNase P RNAs by UV melting, with the goal of optimizing secondary and tertiary structure for crystallization. We demonstrate the utility of this method in two test cases: First, thermodynamic parameters are extracted from the kissing complex, a typical tertiary interaction found in many RNAs. This leads to the discovery that the ionic strength dependence may give clues about the structural transitions observed. Second, we study the effect of mutations on a more complex RNA, the IRES site of hepatitis C virus, and correlate UV melting data to a dynamic equilibrium between alternative structures. The lessons learned from these experiments are then applied to RNase P to disrupt specific tertiary interactions to facilitate crystallization. The effect of these mutations is investigated using both UV melting and a novel, fluorescence-based approach. Finally, crystallization screens are constructed using methods of experimental design, and a new approach for crystallization in gels is developed.
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: Naoki Sugimoto
Publisher: John Wiley & Sons
ISBN: 3527817867
Category : Science
Languages : en
Pages : 288
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Book Description
Discover the fundamentals and intricacies of a subject at the interface of chemistry and biology with this authoritative resource Chemistry and Biology of Non-canonical Nucleic Acids delivers a comprehensive treatment of the chemistry and biology of non-canonical nucleic acids, including their history, structures, stabilities, properties, and functions. You'll learn about the role of these vital compounds in transcription, translation, regulation, telomeres, helicases, cancers, neurodegenerative diseases, therapeutic applications, nanotechnology, and more. An ideal resource for graduate students, researchers in physical, organic, analytical, and inorganic chemistry will learn about uncommon nucleic acids, become the common non-canonical nucleic acids that fascinate and engage academics and professionals in private industry. Split into 15 chapters covering a wide range of aspects of non-canonical nucleic acids, the book explains why these compounds exist at the forefront of a new research revolution at the intersection of chemistry and biology. Chemistry and Biology of Non-canonical Nucleic Acids also covers a broad range of topics critical to understanding these versatile and omnipresent chemicals, including: * A discussion of the dynamic regulation of biosystems by nucleic acids with non-canonical structures * The role played by nucleic acid structures in neurodegenerative diseases and various cancers * An exploration of the future outlook for the chemistry and biology of non-canonical nucleic acids * An introduction to the history of canonical and non-canonical structures of nucleic acids * An analysis of the physicochemical properties of non-canonical nucleic acids Perfect for biochemists, materials scientists, and bioengineers, Chemistry and Biology of Non-canonical Nucleic Acids will also earn a place in the libraries of medicinal and pharmaceutical chemists who wish to improve their understanding of life processes and the role that non-canonical nucleic acids play in them.
Author:
Publisher:
ISBN:
Category : Dissertations, Academic
Languages : en
Pages : 924
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Author: Jens Kurreck
Publisher: Royal Society of Chemistry
ISBN: 0854041168
Category : Medical
Languages : en
Pages : 362
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Book Description
This book provides a compelling overall update on current status of RNA interference
