Download or read book High Resolution Single molecule Enzyme Dynamics Using Nanopores written by Jonathan M. Craig and published by . This book was released on 2017 with total page 139 pages. Available in PDF, EPUB and Kindle. Book excerpt: DNA is a molecule that contains the genetic information of all living organisms. DNA provides the instructions that the cell uses to construct the proteins which carry out the complex functions required for life to thrive. Enzymes are a class of proteins that use chemical potentials to catalyze energetically unfavorable chemical reactions to perform tasks ranging from muscle contraction to DNA packaging. In this thesis I focus on a class of enzymes called `motor enzymes' which use the energy provided by ATP hydrolysis to produce directed motion along a molecular track, such as DNA or RNA, and perform mechanical tasks such as unwinding double-stranded nucleic acids or building double stranded nucleic acids from single-stranded nucleic acids. Classically, enzyme reactions were studied by biochemical methods that monitor a large number of reactions simultaneously. These methods are limited because enzymes operate near thermal energies, leading to asynchronous progression of the chemical reaction. In the past 30 years, methods to monitor the reactions of single enzyme molecules have provided numerous insights into the function of motor enzymes, but these techniques lack the resolution to provide full details of how these molecules transduce chemical energy into mechanical work. In this thesis I present the development of Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), a method for monitoring the movement of single enzyme molecules on DNA at unprecedented spatiotemporal resolution using the biological nanopore MspA. In SPRNT, a single MspA protein pore (termed a `nanopore') in a phospholipid bilayer forms the only electrical connection between two salt solutions termed \textit{cis} and \textit{trans}. A voltage applied across the membrane causes an ion current to flow through the nanopore. Negatively charged single-stranded DNA complexed to a motor enzyme is attracted into the nanopore by the electric field. The DNA passes through the pore until the motor enzyme, which is too large to fit through the pore, comes to rest on the rim of MspA. The DNA bases in the pore reduce the ion current flowing through the pore depending on the bases therein. The motor enzyme then moves along the DNA, causing DNA to move through the pore, leading to a series of stochastic ion-current amplitudes which simultaneously provide measurements of the kinetics of the enzyme and the DNA sequence. This method leads to a higher spatiotemporal resolution than any other single-molecule technique. In this thesis I present my role in the development of SPRNT. In chapter 1 I introduce the relevant biomolecules and techniques used to examine them. In chapter 2 I discuss the development of SPRNT and quantify its spatiotemporal resolution. In chapters 3 and 4 I present the first enzyme dynamics studies done with SPRNT on the helicase Hel308, and use information from quantities that could not be measured previously to elucidate the precise details of Hel308 motion on DNA, and to determine the mechanism by which DNA modulates Hel308 translocation. Chapter 5 contains concluding remarks and a discussion of the future of SPRNT.

Download or read book Single Molecule Enzymology Nanomechanical Manipulation and Hybrid Methods written by and published by Academic Press. This book was released on 2017-01-03 with total page 486 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-Molecule Enzymology, Part B, 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, and nanopore 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, and nanopore and tethered particle motion

Download or read book Single Molecule Enzymology Fluorescence Based and High Throughput Methods written by and published by Academic Press. This book was released on 2016-10-28 with total page 618 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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

Download or read book Polymer Translocation written by M. Muthukumar and published by CRC Press. This book was released on 2016-04-19 with total page 362 pages. Available in PDF, EPUB and Kindle. Book excerpt: Polymer translocation occurs in many biological and biotechnological phenomena where electrically charged polymer molecules move through narrow spaces in crowded environments. Unraveling the rich phenomenology of polymer translocation requires a grasp of modern concepts of polymer physics and polyelectrolyte behavior. Polymer Translocation discusse

Download or read book The Study of Single Molecule Protein Biophysics Using a Solid State Nanopore written by Kevin J. Freedman and published by . This book was released on 2013 with total page 442 pages. Available in PDF, EPUB and Kindle. Book excerpt: The kinetics of protein folding and binding has been studied for several decades and continues to reveal links to overall cellular health,cell functionality, and responses to therapeutic agents. Despite numerous methods to purify and detect proteins, there is still a growing need to develop technology that can enhance sensing and reveal potentially hidden properties of proteins. This is important not only from a scientific perspective but also in practically achieving the goals of personalized healthcare. Next generation sensors should ideally have three main characteristics: (1) high resolution sensing, (2) high-throughput sensing, and (3) the potential to be automated and used by untrained personnel. Future devices will revolutionize the healthcare industry by decreasing both the time and cost to do basic scientific research, diagnostic testing, and drug development. A likely candidate for next-generation protein sensing is solid-state nanopores. The pores developed here are fabricated in a 50 nm thick silicon nitride membrane and a single nanopore is drilled using a focused ion beam or a focused electron beam. The detection method employed is largely based on resistive pulse sensing where analytes are electrokinetically transported through a pore and identified by their unique modulation of ionic current (i.e. an ionic blockade). Since the dimensions of the nanopore are on the same scale as the molecule being sensed, only a single molecule can enter the pore allowing individual protein kinetics to be probed. Traditionally proteins are detected by ensemble averaging which hides important kinetics and sub-populations of molecules that may be important to understanding the early stages of a disease or detect a disease early. In the first section of this study, the prominent issue of protein adsorption onto the sensing device (i.e. the nanopore) is addressed and resolved by using a modified voltage protocol. The rationale behind the new sensing scheme is explained in terms of the interplay of diffusive and entropic (barrier-dominated) forces on a protein. In the second section, we discovered that the voltage which drives the protein through the pore also has denaturing effects. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein. In the final section of this study, voltage-mediated unbinding of a single protein complex is studied. We argue that determining the unbinding forces between two proteins adds an additional level of specificity which is needed for eventual use as a diagnostic tool. In this study, nanopores are developed not only as a sensor but also a single molecule or protein complex manipulator that can locally unfold or unbind molecules.

Download or read book Single Molecule Tools for Bioanalysis written by Shuo Huang and published by CRC Press. This book was released on 2022-05-26 with total page 278 pages. Available in PDF, EPUB and Kindle. Book excerpt: In the last three decades, the fast development of single-molecule techniques has revolutionized the way we observe and understand biological processes. Some of these techniques have been further adapted as tools for bioanalysis. This book summarizes and details the frontiers of the development of these tools as well as their applications. The contributors are young and established researchers in their respective fields. The main content originates from the lecture notes of a chemistry graduate course taught by the book editor at Nanjing University. This book is suitable to be used as a textbook for a high-level undergraduate or an entry-level graduate course. The systematically written content provides a thorough illustration of the mechanisms of each methodology presented.

Download or read book High Resolution Measurements of RNA Polymerase with Nanopore Tweezers written by Ian C. Nova and published by . This book was released on 2020 with total page 164 pages. Available in PDF, EPUB and Kindle. Book excerpt: RNA polymerase (RNAP) is the molecular machine responsible for transcription, the process of making RNA from a DNA template. While the overall role of RNAP in this central cellular process is clear (RNAP moves along double stranded DNA, extending an RNA chain by 1 base with each step), questions remain about the timing and order of events within each reaction cycle [1]. With bulk biochemical assays that average over an ensemble of molecules, determining the dynamics of individual RNAP enzymes during transcription is difficult. For this reason, experiments that capture and observe single RNAP molecules have been particularly elucidating. Specificacally, optical tweezers experiments, in which the position of single RNAP molecules along DNA are tracked over time, have helped develop and test various kinetic models for RNAP transcription [2]. By varying applied force and nucleotide triphosphate (NTP, the substrate for transcription) concentration, these experiments probe the energy landscape and reveal the coupling between chemical hydrolysis and mechanical motion [3]. However, in physiologically relevant conditions, single steps of RNAP occur too quickly and over too small a distance (1 base pair) to be detected by optical tweezers [4]. This necessitates averaging over multiple steps in the reaction cycle, prohibiting direct observation of individual enzyme states. In addition, although particular DNA sequences are known to interact specifically with RNAP, optical tweezers lacks the exact sequence registration, limiting methods of studying sequence - enzyme interactions at high resolution. At the University of Washington, our group has helped develop a new single-molecule technology uniquely suited to study the details of RNAP dynamics. This method, Single molecule Picometer Resolution Nanopore Tweezers (or SPRNT), is based off of the concept of nanopore DNA sequencing [5] and allows monitoring the translocation of single motor enzymes along DNA at unprecedented spatiotemporal resolution (40 picometers at millisecond time scales). In addition, each enzyme position measurement with SPRNT corresponds to a particular DNA sequence, allowing direct observation of enzyme interactions with DNA sequence. SPRNT has already been used to study motor proteins that walk along DNA: a DNA polymerase [6] [7] and a DNA helicase [8] [9]. In this thesis, I present my role in developing SPRNT to investigate RNAP during transcription. In section 0.1, I outline the relevant molecular systems and the techniques used to investigate them. I describe the development of SPRNT, starting with the fundamentals of nanopore sequencing, and compare this new technique to other methods. In section 0.2, I detail the methods developed to investigate E. coli RNAP with SPRNT, providing a guideline for future investigations of this enzyme. In section 0.3, I describe the initial results that were used to motivate and improve upon these methods. In section 0.4, I recount the relevant results of this investigation. I present the first measurements of single steps of E. coli RNAP during transcription at biologically relevant [NTPs] under an assisting force. I summarize how these measurements were used to track enzyme transitions between different states, developing a model for transcription. Using this model, I extrapolate the results obtained under various assisting forces to calculate the relevant rate constants at zero-force, providing a view into how RNAP behaves in its native environment. Next, I describe more SPRNT experiments that investigate RNAP pausing at particular sequences, including the Ơ̐1rst single-molecule detection of a 0half-translocated0 state during pausing. I detail a model for RNAP pausing resulting from this data. Finally, in section 0.5, I discuss the conclusions drawn from this work and the role SPRNT can play in future studies of transcription.

Download or read book Single Molecule Tools for Bioanalysis written by Shuo Huang and published by CRC Press. This book was released on 2022-05-25 with total page 238 pages. Available in PDF, EPUB and Kindle. Book excerpt: In the last three decades, the fast development of single-molecule techniques has revolutionized the way we observe and understand biological processes. Some of these techniques have been further adapted as tools for bioanalysis. This book summarizes and details the frontiers of the development of these tools as well as their applications. The contributors are young and established researchers in their respective fields. The main content originates from the lecture notes of a chemistry graduate course taught by the book editor at Nanjing University. This book is suitable to be used as a textbook for a high-level undergraduate or an entry-level graduate course. The systematically written content provides a thorough illustration of the mechanisms of each methodology presented.

Download or read book De Novo Design of Nanopore like Architectures and Applications of Nanopores to Investigate the SARS CoV 2 Helicase written by Sinduja Karl Marx and published by . This book was released on 2021 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein nanopores form nanometer-sized holes in a lipid bilayer and are widely used as analytical tools to study the flow of ions and other target molecules through them. Recent advancements in nanopore technologies have enabled use of nanopores for nucleic acid sequencing, peptide or protein analysis, and small-molecule identification and filtration. Custom designed nanopores are a promising area of research that may lead to increased accuracy and specificity of nanopore assays, or enable assays that are not possible or otherwise unknown with the existing library of protein nanopores. Designing protein structures de novo requires two steps: designing protein backbones that will result in the desired function, and identifying amino acid sequences that will fold into the desired backbones at their lowest energy states. In chapter 1, I introduce the geometrical and energetic considerations in building protein backbones, with a central channel and a defined sensing region, for the membrane. Using the Rosetta framework, I present de novo design strategies of oligomeric aqueous alpha-helical channels around an ion-channel filter and transmembrane beta-barrels. I show the results of biochemical validation of hexameric aqueous alpha-helical channels, and the ion-conductance properties of the first de novo designed 8-stranded transmembrane beta-barrel. This work is a step towards reducing our dependence on naturally occurring ion-channels that are challenging to modify, and towards creating entirely de novo nanopores with an un-naturally optimal fit for their targets. In chapter 2, I present the application of a naturally occuring nanopore to investigate the helicase found in Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nonstructural protein 13 (nsp 13) helicase is involved in the replication of the SARS-CoV-2 genome and a potential drug target for antivirals. Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) is a new technique that uses MspA, a biological nanopore, to monitor the movement of single enzyme molecules on DNA at high spatiotemporal resolution. Using SPRNT, I present the first single-molecule assay to monitor the kinetics of single-nucleotide steps of the SARS-CoV-2 helicase, nsp13. This assay provides a single-molecule platform for understanding how antiviral compounds affect nsp13 function and glean insights for their development.

Download or read book Getting the Most Out of Nanopores written by Henry Brinkerhoff and published by . This book was released on 2019 with total page 254 pages. Available in PDF, EPUB and Kindle. Book excerpt: Over the past three decades, the fields of biophysics and biotechnology have seen an era of unprecedented growth, bolstered by the development of new experimental techniques. Prominent within these techniques are “single-molecule” methods which enable the observation and manipulation of single biomolecules. These techniques allow for controlled experiments on the most fundamental structures composing life. The function of living organisms is governed by statistical physics, and traditional bulk chemical methods report only an average of the rich and heterogeneous activity of these biological structures. Therefore, bulk methods provide an incomplete picture of the mechanical behavior of biomolecules, and of the way life stores, modifies and accesses information. Early single-molecule experiments using flow cells and magnetic tweezers were initially used to study the passive mechanics of molecules like DNA and the behav- ior of stepping enzymes including myosin and kinesin. These enzyme experiments demonstrated the power of single-molecule techniques, showing that enzyme behavior is fundamentally statistical, moving randomly with a slight rectification provided by chemical potentials maintained by the cell. Soon thereafter, an expanding repertoire of single-molecule methods including superresolution micrscopy, single-fluorophore microscopy and optical tweezers refined and expanded these results, making plain the diversity and complexity of the mechanical behavior of biomolecules. Advances in genomics over this time period made it clear that the nucleic acids DNA and RNA, which store the information passed down and used by life to encode the sequences of every protein it produces, are also subject to the statistical physics governing biomolecules. Damage to DNA and its repair, the formation of secondary structure, the insertion of viral DNA fragments, replication, recombination, modifica- tion of bases, and regulation of gene expression: these are all fundamentally random processes. As recording and analyzing vast amounts of data has become more feasible with access to greater computing power, it has become clear that methods sequencing only large samples of many DNA molecules fail to recognize the variance crucial to the functionality of life’s genetic library. The scientific appeal of a single-molecule sequencing technique together with a push for longer read lengths and cheaper sequencing led to the development of nanopore sequencing, a method using a nanometer-scale hole in a thin membrane to trap and analyze DNA. Beginning with the demonstration of nanopores as single molecule “Coulter counters,” through results proving that nanopore experiments can discriminate between trapped DNA strands with different base content, we now have arrived in an era where nanopores are used in commercial DNA sequencing platforms and high-precision single molecule biophysics experiments. Within this dissertation, I provide a “user manual” of sorts for collecting and understanding the single-molecule information provided by nanopore experiments. Then, through two examples of concrete improvements to the nanopore DNA se- quencing system, I demonstrate how a thorough understanding and adequate physi- cal model of the system can motivate experiment and invention. My hope is that a scientist wishing to perform nanopore experiments for the first time will find this to be a useful guide for executing the experiments, as well as for modeling and analyzing the rich and complex signals that they generate. In part I, background is provided on the arena in which these experiments play out. I first introduce key properties of DNA and other biological molecules, as well as the history and future of DNA sequencing. Part II contains a guide to the experimental setup and operation of a nanopore experiment. I also delve deeper into the biophysics of the experiment as it is performed at the University of Washington, discussing properties of the enzyme-DNA-nanopore complex, and I discuss the signal obtained from the experiment and its properties. In part IV, describe the ways the nanopore signal can be modeled, reduced, an- alyzed, and interpreted, including introductions to some commonplace analysis tools used to study single molecule data. Finally, part IV shows how using the results of this model, we developed extensions and modifications of the nanopore experiment, improve the accuracy and flexibility of nanopore DNA sequencing. My work at the University of Washington is included primarily in part IV, much of which I completed in collaboration with primarily Dr. Andrew Laszlo and Dr. Brian Ross, and IV, in which the variable-voltage experiments were completed in collaboration with Dr. Matthew Noakes.

Download or read book Single Molecule Sensing Beyond Fluorescence written by Warwick Bowen and published by Springer Nature. This book was released on 2022-03-01 with total page 426 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides an interesting snapshot of recent advances in the field of single molecule nanosensing. The ability to sense single molecules, and to precisely monitor and control their motion is crucial to build a microscopic understanding of key processes in nature, from protein folding to chemical reactions. Recently a range of new techniques have been developed that allow single molecule sensing and control without the use of fluorescent labels. This volume provides an overview of recent advances that take advantage of micro- and nanoscale sensing technologies and provide the prospect for rapid future progress. The book endeavors to provide basic introductions to key techniques, recent research highlights, and an outlook on big challenges in the field and where it will go in future. It is a valuable contribution to the field of single molecule nanosensing and it will be of great interest to graduates and researchers working in this topic.

Download or read book Confining Electrochemistry to Nanopores written by Yi-Tao Long and published by Royal Society of Chemistry. This book was released on 2020-10-26 with total page 267 pages. Available in PDF, EPUB and Kindle. Book excerpt: The nanopore provides a highly electrochemically confined space within which single-molecule characteristics can be efficiently converted into measurable electrochemical signatures with high temporal and current resolution. Aimed at developing the concept of the electrochemical confined space in analysing single molecules, this book serves as a stepping-stone to many exciting discoveries in nanopore-based analysis of biological processes and chemical reactions in confined space. The field of nanopore sensors is growing rapidly, but there have been no new books on nanopore technology that provide an overview of the research on nanopore-based sensing until now. The book provides a good source of nanopore studies for researchers interested in and working in the general areas of electrochemistry and nanobiotechnology, especially on nanopore sensors.

Download or read book Single Molecule Spectroscopy written by R. Rigler and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 375 pages. Available in PDF, EPUB and Kindle. Book excerpt: The topics range from single molecule experiments in quantum optics and solid-state physics to analogous investigations in physical chemistry and biophysics.

Download or read book Protein Interactions written by Jianfeng Cai and published by BoD – Books on Demand. This book was released on 2012-03-16 with total page 478 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein interactions, which include interactions between proteins and other biomolecules, are essential to all aspects of biological processes, such as cell growth, differentiation, and apoptosis. Therefore, investigation and modulation of protein interactions are of significance as it not only reveals the mechanism governing cellular activity, but also leads to potential agents for the treatment of various diseases. The objective of this book is to highlight some of the latest approaches in the study of protein interactions, including modulation of protein interactions, development of analytical techniques, etc. Collectively they demonstrate the importance and the possibility for the further investigation and modulation of protein interactions as technology is evolving.

Download or read book Nanofabrication Towards Biomedical Applications written by Challa S. S. R. Kumar and published by John Wiley & Sons. This book was released on 2006-03-06 with total page 442 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book focuses on the materials, synthetic methods, tools and techniques being developed in the nanoregime towards the life sciences -- in particular biology, biotechnology and medicine. Readers from materials science, engineering, chemistry, biology and medical backgrounds will find detailed accounts of the design and synthesis of nanomaterials and the tools and techniques involved in their production for applications in biology, biotechnology and medicine.

Download or read book Microsecond Dynamics of Enzymes written by Maxim V. Akhterov and published by . This book was released on 2015 with total page 104 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book DNA polymerases in Biotechnology written by Zvi Kelman and published by Frontiers Media SA. This book was released on 2015-03-18 with total page 147 pages. Available in PDF, EPUB and Kindle. Book excerpt: DNA polymerases are core tools for molecular biology including PCR, whole genome amplification, DNA sequencing and genotyping. Research has focused on discovery of novel DNA polymerases, characterization of DNA polymerase biochemistry and development of new replication assays. These studies have accelerated DNA polymerase engineering for biotechnology. For example, DNA polymerases have been engineered for increased speed and fidelity in PCR while lowering amplification sequence bias. Inhibitor resistant DNA polymerase variants enable PCR directly from tissue (i.e. blood). Design of DNA polymerases that efficiently incorporate modified nucleotide have been critical for development of next generation DNA sequencing, synthetic biology and other labeling and detection technologies. The Frontiers in Microbiology Research Topic on DNA polymerases in Biotechnology aims to capture current research on DNA polymerases and their use in emerging technologies.