Combined Optical Trapping and Sm-FRET for Nucleic Acid Folding Studies PDF Download
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Author: Christian Francisco Perez
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
By studying ancient RNA motifs found in all kingdoms of life, we are also looking back in time to a common evolutionary ancestor. Riboswitches and ribozymes are structured RNAs capable of gene regulation and catalysis. Their surprising catalytic/functional diversity and prevalence across species hints at an "RNA World" that predates our current paradigm which depends on DNA for information and protein for catalysis. The TPP riboswitch is an ancient ligand-binding RNA that regulates vitamin B1 (TPP) synthesis in bacteria, archaea, and even higher organisms like plants. Using single molecule optical trapping (OT) and the judicious application of piconewton forces, we have studied the folding pathways of the riboswitch and its response to TPP and other analogues. Ligand binding appears to form in two steps: TPP must first bind weakly before proceeding to a strongly bound state, as measured by the non-equilibrium work required to unfold it. To probe the physical origin of these states, we use a novel dual-beam optical trap combined with simultaneous FRET that can measure two orthogonal signals of the molecule's conformation. We demonstrate the direct observation of both the unfolding of secondary (base-paired) structures (via OT) and long-range tertiary contacts (via FRET) that form during ligand binding. In time, we anticipate this technique will become a sought-after tool that can expose the intricate folding pathways of complex biomolecules.
Author: Christian Francisco Perez
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
By studying ancient RNA motifs found in all kingdoms of life, we are also looking back in time to a common evolutionary ancestor. Riboswitches and ribozymes are structured RNAs capable of gene regulation and catalysis. Their surprising catalytic/functional diversity and prevalence across species hints at an "RNA World" that predates our current paradigm which depends on DNA for information and protein for catalysis. The TPP riboswitch is an ancient ligand-binding RNA that regulates vitamin B1 (TPP) synthesis in bacteria, archaea, and even higher organisms like plants. Using single molecule optical trapping (OT) and the judicious application of piconewton forces, we have studied the folding pathways of the riboswitch and its response to TPP and other analogues. Ligand binding appears to form in two steps: TPP must first bind weakly before proceeding to a strongly bound state, as measured by the non-equilibrium work required to unfold it. To probe the physical origin of these states, we use a novel dual-beam optical trap combined with simultaneous FRET that can measure two orthogonal signals of the molecule's conformation. We demonstrate the direct observation of both the unfolding of secondary (base-paired) structures (via OT) and long-range tertiary contacts (via FRET) that form during ligand binding. In time, we anticipate this technique will become a sought-after tool that can expose the intricate folding pathways of complex biomolecules.
Author: Jeffrey Robert Vieregg
Publisher:
ISBN:
Category :
Languages : en
Pages : 468
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Author: Paul R. Selvin
Publisher: CSHL Press
ISBN: 087969775X
Category : Science
Languages : en
Pages : 511
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Book Description
Geared towards research scientists in structural and molecular biology, biochemistry, and biophysics, this manual will be useful to all who are interested in observing, manipulating and elucidating the molecular mechanisms and discrete properties of macromolecules.
Author:
Publisher: Academic Press
ISBN: 0128095474
Category : Science
Languages : en
Pages : 618
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Book Description
Single-Molecule Enzymology, Part A, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in single-molecule enzymology, and includes sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, nanopores, and tethered particle motion. - Continues the legacy of this premier serial with quality chapters authored by leaders in the field - Covers research methods in single-molecule enzymology - Contains sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, nanopores, and tethered particle motion
Author:
Publisher: Academic Press
ISBN: 0123814839
Category : Science
Languages : en
Pages : 745
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Book Description
Single molecule tools have begun to revolutionize the molecular sciences, from biophysics to chemistry to cell biology. They hold the promise to be able to directly observe previously unseen molecular heterogeneities, quantitatively dissect complex reaction kinetics, ultimately miniaturize enzyme assays, image components of spatially distributed samples, probe the mechanical properties of single molecules in their native environment, and "just look at the thing" as anticipated by the visionary Richard Feynman already half a century ago. Single Molecule Tools, Part B: Super-Resolution, Particle Tracking, Multiparameter, and Force Based Methods captures a snapshot of this vibrant, rapidly expanding field, presenting articles from pioneers in the field intended to guide both the newcomer and the expert through the intricacies of getting single molecule tools. - Includes time-tested core methods and new innovations applicable to any researcher employing single molecule tools - Methods included are useful to both established researchers and newcomers to the field - Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines
Author:
Publisher: Elsevier
ISBN: 0124116272
Category : Science
Languages : en
Pages : 368
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Book Description
Published continuously since 1944, Advances in Protein Chemistry and Structural Biology has been a continuous, essential resource for protein chemists. Covering reviews of methodology and research in all aspects of protein chemistry, including purification/expression, proteomics, modeling and structural determination and design, each volume brings forth new information about protocols and analysis of proteins while presenting the most recent findings from leading experts in a broad range of protein-related topics. - Covers reviews of methodology and research in all aspects of protein chemistry - Brings forth new information about protocols and analysis of proteins while presenting the most recent findings from leading experts in a broad range of protein-related topics
Author: Cho-Ying Chuang
Publisher:
ISBN: 9780438864726
Category : Electronic dissertations
Languages : en
Pages : 155
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Book Description
Nucleic acids and proteins are fundamental molecular units of life. To understand their properties, we need powerful tools that allow investigation at the single-molecule level. Over the past three decades, the development of single-molecule force and fluorescence techniques has provided us new knowledge that was previously unattainable through ensemble measurements. However, we lack methods that allow us to precisely measure the mechanical properties of these molecules while visually detecting multiple molecules at the same time. In this dissertation, we maximize the information obtained in single-molecule measurements by pushing the techniques to be more precise and more complex. We then utilize our instrument to directly observe the folding and unfolding of the nucleic acid G-quadruplex structure. Among the single-molecule force techniques, angstrom-resolution has been achieved by optical tweezers using the dual-trap instrument design. Dual-traps can be produced by acousto-optic (AO) devices, which have many advantages, but trap positioning inaccuracies have limited their usage at high-resolution. We have designed a method to remove the inaccuracies by randomizing the phase of the radio frequency that drives the AO device. We demonstrated that the trap inaccuracies are completely eliminated and high-resolution trapping quality is achieved. This advance allows us to perform long duration measurements with reduced drift in trap measurement over time. Next, we present instrumentation advances that combine high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy along with automated single molecule assembly. Multicolor not only allows the detection of multiple observables but also increases the flexibility in the choice of fluorophores. We demonstrated the ability to simultaneously measure angstrom-scale changes in tether extension and single fluorophore signals. The biggest challenge in integrating optical tweezers and fluorescence is the potential for greatly enhanced photobleaching which can make experiments impossible. We showed that the mean number of photons emitted before bleaching is unaffected by the trap laser when interlacing the fluorescence and optical trap lasers. We investigated the photostability of quantum dots and fluorophores. Finally, we devised computer-controlled automation to conserve the fluorophore lifetime. This advance enables us to observe multiple molecules or multiple degrees of freedom within a molecular complex while mechanically manipulate and detect them. Taking advantage of these method and instrumentation advances, we investigate the folding and unfolding of a DNA secondary structure: thrombin-binding aptamer G-quadruplex (TBA-GQ). Studying the kinetics of G-quadruplex formation is essential for understanding telomere regulation (the ends of chromosomes) and therapeutic approaches for disease. TBA-GQ is the smallest G-quadruplex. Although many experiments and simulations have been done on G-quadruplex, the small size and low stability make it very difficult to observe folding and unfolding of TBA-GQ directly. Our high-resolution optical tweezers have the sensitivity and stability to directly observe TBA-GQ at very low forces. We found that with increasing force, the folding rate decreased and the unfolding rate increased. Our work demonstrates that at a given force, the TBA-GQ formation is facilitated by metal ions and is stabilized by thrombin. It also indicates that the equilibrium force increased as KCl concentration increased. From a detailed analysis of the folding and unfolding rate constants vs applied force, we were able to detect a single transition state conserved across all conditions and identify the structure of the transition state as the G-triplex structure.
Author: Peter Hinterdorfer
Publisher: Springer Science & Business Media
ISBN: 0387764976
Category : Science
Languages : en
Pages : 634
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Book Description
This handbook describes experimental techniques to monitor and manipulate individual biomolecules, including fluorescence detection, atomic force microscopy, and optical and magnetic trapping. It includes single-molecule studies of physical properties of biomolecules such as folding, polymer physics of protein and DNA, enzymology and biochemistry, single molecules in the membrane, and single-molecule techniques in living cells.
Author: Daniel William Hogan
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
This dissertation covers three research projects, the instrumentation needed to conduct them, and the physics which underlie the techniques. All projects employ optical tweezers as a primary method, but are otherwise quite diverse: two are strictly in vitro experiments, while one includes extensive computer simulation; two involve protein, while the third considers a nucleic acid system; two employ single-molecule FRET to measure an additional reaction coordinate, while one gains its data solely from a single-beam optical trap. Homodimeric KIF17 and heterotrimeric KIF3AB are processive, kinesin-2 family motors that act jointly to carry out anterograde IFT, ferrying cargo along MTs toward the tips of cilia. How IFT trains attain speeds that exceed the unloaded rate of the slower, KIF3AB motor remains unknown. By characterizing the motility properties of kinesin-2 motors as a function of load we find that the increase in KIF3AB velocity, elicited by forward loads from KIF17 motors, cannot alone account for the speed of IFT trains in vivo. Instead, higher IFT velocities arise from an increased likelihood that KIF3AB motors dissociate from the MT, resulting in transport by KIF17 motors alone, unencumbered by opposition from KIF3AB. The rate of transport is therefore set by an equilibrium between a faster state, where only KIF17 motors move the train, and a slower state, where at least one KIF3AB motor on the train remains active in transport. The more frequently the faster state is accessed, the higher the overall velocity of the IFT train. We conclude that IFT velocity is governed by the absolute numbers of each motor type on a given train, how prone KIF3AB is to dissociation from MTs relative to KIF17, and how prone both motors are to dissociation relative to binding MTs. The TPP riboswitch is a cis-regulatory element in mRNAs that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and FRET, using an optical tweezers instrument equipped for simultaneous single-molecule FRET. The "force-FRET" approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in single-molecule FRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity. Hairpins in nascent RNAs extend RNAP pause durations, but the mechanism of stabilization is poorly understood. It was speculated that a conformational change in the RNAP clamp induced by the formation of the hairpin in the RNA exit channel was responsible. Using a dual-beam optical tweezers instrument with single-molecule FRET detection capabilities, we examine the conformation of the clamp during elongation, elemental pauses, hairpin-stabilized pauses, and termination pauses. We find that there is only negligible change in the clamp conformation upon entry to a hairpin-stabilized pause, with a displacement an order of magnitude small than had been predicted. Based on our and prior results, we speculate as to other possible mechanisms. In the course of this work, I developed a full suite of applications to efficiently control and collect data from a modern optical tweezers instrument, which adheres to best practices for realtime data collection; it can now be freely downloaded.
Author: Dongyou Liu
Publisher: CRC Press
ISBN: 1040005640
Category : Science
Languages : en
Pages : 763
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Book Description
With a history that likely dates back to the dawn of human civilization more than 10,000 years ago, and a record that includes the domestication and selective breeding of plants and animals, the harnessing of fermentation process for bread, cheese, and brewage production, and the development of vaccines against infectious diseases, biotechnology has acquired a molecular focus during the 20th century, particularly following the resolution of DNA double helix in 1953, and the publication of DNA cloning protocol in 1973, and transformed our concepts and practices in disease diagnosis, treatment and prevention, pharmaceutical and industrial manufacturing, animal and plant industry, and food processing. While molecular biotechnology offers unlimited opportunities for improving human health and well-being, animal welfare, agricultural innovation and environmental conservation, a dearth of high quality books that have the clarity of laboratory manuals without distractive procedural details and the thoroughness of well-conversed textbooks appears to dampen the enthusiasm of aspiring students. In attempt to fill this glaring gap, Handbook of Molecular Biotechnology includes four sections, with the first three presenting in-depth coverage on DNA, RNA and protein technologies, and the fourth highlighting their utility in biotechnology. Recognizing the importance of logical reasoning and experimental verification over direct observation and simple description in biotechnological research and development, the Introduction provides pertinent discussions on key strategies (i.e., be first, be better, and be different), effective thinking (lateral, parallel, causal, reverse, and random), and experimental execution, which have proven invaluable in helping advance research projects, evaluate and prepare research reports, and enhance other scientific endeavors. Key features Presents state-of-the-art reviews on DNA, RNA and protein technologies and their biotechnological applications Discusses key strategies, effective thinking, and experimental execution for scientific research and development Fills the gap left by detailed-ridden laboratory manuals and insight-lacking standard textbooks Includes expert contributions from international scientists at the forefront of molecular biotechnology research and development Written by international scientists at the forefront of molecular biotechnology research and development, chapters in this volume cover the histories, principles, and applications of individual techniques/technologies, and constitute stand-alone, yet interlinked lectures that strive to educate as well as to entertain. Besides providing an informative textbook for tertiary students in molecular biotechnology and related fields, this volume serves as an indispensable roadmap for novice scientists in their efforts to acquire innovative skills and establish solid track records in molecular biotechnology, and offers a contemporary reference for scholars, educators, and policymakers wishing to keep in touch with recent developments in molecular biotechnology.
