DOCSLIB.ORG

Riboswitch-Based Sensors of Small Molecules in Bacteria

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Riboswitch-Based Sensors of Small Molecules in Bacteria RIBOSWITCH-BASED SENSORS OF SMALL MOLECULES IN BACTERIA THE ENGINEERING OF RIBOSWITCH-BASED SENSORS OF SMALL MOLECULES IN BACTERIA AND THEIR APPLICATION IN THE STUDY OF VITAMIN B12 BIOLOGY By CASEY C. FOWLER B.Sc., Queen’s University, Kingston Ontario, 2004 A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy McMaster University © by Casey C. Fowler, August 2011 DOCTOR OF PHILOSOPHY (2011) McMaster University (Biochemistry & Biomedical Sciences) Hamilton, Ontario TITLE: The Engineering of Riboswitch-Based Sensors of Small Molecules in Bacteria and Their Application in the Study of Vitamin B12 Biology AUTHOR: Casey C. Fowler, B.Sc. (Queen’s University) SUPERVISOR: Dr. Yingfu Li NUMBER OF PAGES: xiii, 191 ii Abstract Small molecule metabolites have important and diverse roles in every major cellular function. To study the activities of metabolites and the biological processes in which they are involved, it is important to be able to detect their levels within cells. Technologies that measure the concentrations of small molecules within the context of living, growing cells are highly advantageous but are challenging to produce. In this thesis, a novel class of intracellular small molecule sensors is produced, characterized and applied to address novel and relevant research questions. These sensors detect a specific target molecule within bacterial cells using RNA regulatory elements known as riboswitches and one of many possible reporter proteins. In addition to a project that yielded new methodology to create custom riboswitches, two projects that assess the capabilities of sensors that detect an active form of vitamin B12 are described. These projects present an abundance of data that provide novel insights into the transport and metabolism of vitamin B12 in E. coli cells. Overall, the results presented indicate that riboswitch-based sensors represent valuable and unique tools for the study of microbial biology. The thesis is concluded with a discussion that describes design strategies and several exciting potential applications for future riboswitch sensors. iii Acknowledgements First and foremost I would like to thank my supervisors Dr. Yingfu Li and Dr. Eric Brown. Their contributions to the research presented in this thesis and to my development as a scientist have been monumental. My interactions with both of these fantastic mentors will continue to benefit me for the remainder of my career. I would also like to recognize the contributions of my other supervisory committee member, Dr. David Andrews. His vigorous and carefully considered questions and suggestions kept me on my toes during committee meetings and were very helpful in addressing problems that arose over the course of my research. I would also like to acknowledge Dr. Murray Junop for the many long discussions we’ve had regarding the work described in Chapter 5 of this thesis. With his guidance I have learned a great deal about structural biology. The Li lab has provided a pleasant and productive research environment throughout my time at McMaster. I would like to recognize Simon Mcmanus, Naveen Kumar, Wendy Mok and Kacper Kuryllo for the useful discussions, advice and assistance they have provided in the lab. On a more personal level, I would like to thank Naomi as well as my family and friends for making my life outside of the lab so enjoyable throughout my time in graduate school. iv To my parents, who have set and kept me on a happy path in life. v Table of Contents Abstract.............................................................................................................................iii Acknowledgements...........................................................................................................iv List of Tables.....................................................................................................................ix List of Figures.....................................................................................................................x List of Abbreviations.......................................................................................................xii Chapter 1. A General Introduction.................................................................................1 1.1 Overview of the problem to be addressed…………..............................................1 1.2 The isolation, properties and applications of aptamers .........................................9 1.3 Riboswitches: metabolite-sensing RNA regulatory elements..............................18 1.4 The engineering and applications of bacterial riboswitches................................35 1.5 Vitamin B12 and its synthesis, uptake and utilization in E. coli cells.................44 1.6 Specific research objectives and thesis organization...........................................52 Chapter 2. A FACS-Based Approach to Engineering Artificial Riboswitches.........57 2.1 Author’s preface……………………………....................................................57 2.2 Abstract...............................................................................................................59 2.3 Introduction........................................................................................................59 2.4 Materials and Methods......................................................................................63 2.4.1 Growth media, strains, plasmids and molecular cloning.............................63 2.4.2 Fluorescence activated cell sorting and colony isolation.............................64 2.4.3 Fluorescence assays.....................................................................................65 vi 2.5 Results.................................................................................................................66 2.5.1 Selection scheme and library design............................................................66 2.5.2 FACS to isolate candidate riboswitch sequences.........................................67 2.5.3 Assaying candidate riboswitches for theophylline response.......................70 2.5.4 Mechanistic characterization of RS11A......................................................72 2.6 Discussion...........................................................................................................75 2.7 References...........................................................................................................78 Chapter 3. Using a Riboswitch Sensor to Examine Coenzyme B12 Metabolism and Transport in E. coli .........................................................................................................80 3.1 Author’s preface……………………………....................................................80 3.2 Abstract...............................................................................................................81 3.3 Introduction........................................................................................................82 3.4 Materials and Methods......................................................................................85 3.4.1 Plasmids and cell strains..............................................................................85 3.4.2 Molecular cloning........................................................................................86 3.4.3 Growth conditions and reporter assays........................................................86 3.4.4 Ethanolamine utilization assays...................................................................88 3.5 Results.................................................................................................................89 3.5.1 Construction of riboswitch-based AdoCbl sensors......................................89 3.5.2 Riboswitch-based sensors can be used to monitor AdoCbl metabolism.....93 3.5.3 Riboswitch-based sensors can detect the availability of B12 lower axial ligands.........................................................................................................................98 3.5.4 Riboswitch-based sensors can be used to monitor AdoCbl transport........100 3.6 Discussion.........................................................................................................103 vii 3.7 References.........................................................................................................107 Chapter 4. Characterization of Intermolecular Interactions Involving the Periplasmic Binding Protein BtuF Using Riboswitch-Based Sensors.......................113 4.1 Author’s preface………………………..........................................................113 4.2 Abstract.............................................................................................................114 4.3 Introduction......................................................................................................115 4.4 Materials and Methods....................................................................................119 4.4.1 Strains, plasmids and molecular cloning...................................................119 4.4.2 Low-throughput reporter assays................................................................120 4.4.3 Reporter assays for saturation mutagenesis libraries.................................122 4.4.4 Ethanolamine utilization assays.................................................................123 4.5 Results...............................................................................................................123
Load more

Recommended publications
  • Fluorescence Imaging of Metal Ions Implicated in Diseases
    Chemical Society Reviews Fluorescence imaging of metal ions implicated in diseases Journal: Chemical Society Reviews Manuscript ID: CS-HIG-09-2014-000292.R1 Article Type: Highlight Date Submitted by the Author: 08-Dec-2014 Complete List of Authors: Qian, Xu Hong; East China University of Science and Technology, Xu, Zhaochao; Key Laboratory of Separation Science for Analytical Chemistry of CAS, Dalian Institute of Chemical Physics,, Chinese Academy of Sciences, Page 1 of 7 CREATED USING THE RSC ARTICLEChemical TEMPLATE (VER. Society 2.0) - SEE WWW.RSC.ORG/ELECTRONICFILESReviews FOR DETAILS [JOURNAL NAME HERE] | www.rsc.org/[JOURNAL] Article Fluorescence Imaging of Metal ions Implicated in Diseases Xuhong Qian*a and Zhaochao Xu*b Receipt/Acceptance Data [DO NOT ALTER/DELETE THIS TEXT] Publication data [DO NOT ALTER/DELETE THIS TEXT] DOI: 10.1039/b000000x [DO NOT ALTER/DELETE THIS TEXT] Metal ions play an important role in various biological processes; their abnormal homeostasis in cells are related to many diseases, such as neurodegenerative disease, cancer and diabetes. Fluorescent imaging offers an unique route to detect metal ions in cells via a contactless and damage-free way with high spatial and temporal fidelity. Consequently, it represents a promsing method to advance the understanding of physiological and pathological functions of metal ions in cell biology. In this highlight article, we will discuss recent advance in fluorescent imaging of metal ions by small-molecule sensors for understanding the role of metals in related diseases. We will also discuss challenges and opportunities for the design of small-molecule sensors for fluorescent detection of cellular metal ions as a potential method for disease diagnosis.
    [Show full text]
  • Faculty Research in Chemistry at UCA.Docx
    Faculty Research in Chemistry at UCA Table of Contents For more information on each professor’s research please see the links below. Analytical Chemistry Dr. Mauldin He is currently working on a project involving Green Chemistry in which molybdenum (Mo) levels in waste from teaching labs is chemically treated and converted to an environmentally-safe form, using atomic absorption spectroscopy to measure Mo levels in treated versus untreated chemical waste. Dr. Yarberry Her primary current interest is in the field of chemical education. Biochemistry Dr. Dunlap Her research is on calcium regulated proteins. Dr. Isom Her research involves the computational analysis of 3D biomacromolecular structures. Dr. Kelley Her research interest is in the field of retinoid metabolism. Inorganic Chemistry Dr. Desrochers His research studies the coordination chemistry of metal ions. Dr. Massey Her research focuses on making molecules to transform carbon dioxide into an alternative fuel source and developing new methods to improve learning chemistry. Dr. Yang His research focuses on the nitrous oxide and carbon dioxide activation and construction of dynamic coordination polymer materials Organic Chemistry Dr. Barnett Her research is focused on the development and design of methods for sustainable catalysis. Dr. Carter His research interests include investigating the role played by free radical intermediates in the damage of biologically important molecules. Dr. Naumiec His research is on the development of drug candidates to treat tropical diseases. Dr. Tarkka The goals of his research project are new methods for protein purification and quantification of sulfur-containing amino acids. Physical Chemistry Dr. Dooley Her research involves the measurement of aerosol optical properties using pulsed laser cavity ring-down spectroscopy.
    [Show full text]
  • Uptake Into Metazoan Cells
    A Sensitive, Nonradioactive Assay for Zn(II) Uptake into Metazoan Cells The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Richardson, Christopher E. R. et al. “A Sensitive, Nonradioactive Assay for Zn(II) Uptake into Metazoan Cells.” Biochemistry 57 (2018): 6807-6815 © 2018 The Author(s) As Published https://dx.doi.org/10.1021/ACS.BIOCHEM.8B01043 Publisher American Chemical Society (ACS) Version Author's final manuscript Citable link https://hdl.handle.net/1721.1/125596 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Biochemistry Manuscript Author . Author manuscript; Manuscript Author available in PMC 2019 December 18. Published in final edited form as: Biochemistry. 2018 December 18; 57(50): 6807–6815. doi:10.1021/acs.biochem.8b01043. A sensitive, non-radioactive assay for Zn(II) uptake into metazoan cells Christopher E. R. Richardson, Elizabeth M. Nolan, Matthew D. Shoulders*, and Stephen J. Lippard* Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States Abstract Sensitive measurements of cellular Zn(II) uptake currently rely on quantitating radioactive emissions from cells treated with 65Zn(II). Here, we describe a straightforward and reliable method employing a stable isotope to sensitively monitor Zn(II) uptake by metazoan cells. First, biological media selectively depleted of natural abundance Zn(II) using A12-resin [Richardson, C. E.
    [Show full text]
  • Visualizing Metal Ions in Cells: an Overview of Analytical Techniques, Approaches, and Probes☆
    Biochimica et Biophysica Acta 1823 (2012) 1406–1415 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr Review Visualizing metal ions in cells: An overview of analytical techniques, approaches, and probes☆ Kevin M. Dean, Yan Qin, Amy E. Palmer ⁎ article info abstract Article history: Quantifying the amount and defining the location of metal ions in cells and organisms are critical steps in un- Received 6 February 2012 derstanding metal homeostasis and how dyshomeostasis causes or is a consequence of disease. A number of Received in revised form 2 April 2012 recent advances have been made in the development and application of analytical methods to visualize metal Accepted 3 April 2012 ions in biological specimens. Here, we briefly summarize these advances before focusing in more depth on Available online 13 April 2012 probes for examining transition metals in living cells with high spatial and temporal resolution using fluores- cence microscopy. This article is part of a Special Issue entitled: Cell Biology of Metals. Keywords: Metal ion homeostasis © 2012 Elsevier B.V. All rights reserved. Fluorescence imaging Fluorescent sensors Imaging metal ions in cells 1. Introduction Transition metals can exist in many different forms within cells, including free ions,1 bound to biomolecules such as proteins, or in as- Metal ions play fundamental roles in biology by serving as essen- sociation with low molecular weight species such as amino acids or tial cofactors in processes such as respiration, growth, gene transcrip- glutathione, from which the metal ion could be released by changes tion, enzymatic reactions, cell proliferation and immune function.
    [Show full text]
  • Thiophene-Based Molecular Sensors Towards the Selective Detection of Mercury(II) and Other Metals
    Portland State University PDXScholar Dissertations and Theses Dissertations and Theses Summer 8-6-2018 Thiophene-Based Molecular Sensors towards the Selective Detection of Mercury(II) and Other Metals Austin Kazuo Shigemoto Portland State University Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds Part of the Chemistry Commons Let us know how access to this document benefits ou.y Recommended Citation Shigemoto, Austin Kazuo, "Thiophene-Based Molecular Sensors towards the Selective Detection of Mercury(II) and Other Metals" (2018). Dissertations and Theses. Paper 4517. https://doi.org/10.15760/etd.6401 This Dissertation is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. Thiophene-Based Molecular Sensors towards the Selective Detection of Mercury(II) and Other Metals by Austin Kazuo Shigemoto A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Dissertation Committee: Theresa McCormick, Chair Robert Strongin Mark Woods Raj Solanki Portland State University 2018 Abstract This work describes several thiophene-based molecular sensors and various modifications aimed to improve and understand the photophysical and supramolecular properties, such as the association constant (Ka) and selectivity, towards the development of a selective mercury(II) sensor. From the first generation of sensors containing pyridine and thiophene groups, it was determined that thiophene can offer good selectivity for mercury(II) against other transition metal ions, and provide a ratiometric absorption and fluorescent response.
    [Show full text]
  • Full Dissertation Grad Division2
    Molecular Imaging Approaches to Understanding the Roles of Copper in Biology By Dylan Wythe Domaille A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Christopher J. Chang, Chair Professor Kenneth N. Raymond Professor Carolyn R. Bertozzi Professor Jhih-Wei Chu Spring 2011 Molecular Imaging Approaches to Understanding the Roles of Copper in Biology © 2011 by Dylan Wythe Domaille Abstract Molecular Imaging Approaches to Understanding the Roles of Copper in Biology by Dylan Wythe Domaille Doctor of Philosophy in Chemistry University of California, Berkeley Professor Christopher J. Chang, Chair Cells exert precise control over their cellular copper pools through a sophisticated array of uptake, trafficking, and storage mechanisms that effectively maintain a low concentration of thermodynamically free copper ions while maintaining excellent kinetic lability of cellular copper stores. In higher eukaryotes and humans, and particularly in specialized cell types associated with brain, heart, intestine, and liver tissue, the molecular specifics of how kinetically labile copper pools are regulated at the subcellular level and the consequences of copper misregulation in aging and disease remain insufficiently understood. Biochemical and genetic studies have established a broad understanding of how cells acquire, maintain, redirect and release copper ions, while also identifying key proteins involved in these activities. The precise role of the copper ion, however, is more difficult to determine, owing mainly to a dearth of methods for directly following the fate of cellular copper stores. This dissertation describes the design, synthesis, and characterization a several new Cu(I)-responsive fluorophores.
    [Show full text]
  • UC Berkeley Electronic Theses and Dissertations
    UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Molecular Imaging Approaches to Understanding the Roles of Copper in Biology Permalink https://escholarship.org/uc/item/73t8x5vh Author Domaille, Dylan Publication Date 2011 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Molecular Imaging Approaches to Understanding the Roles of Copper in Biology By Dylan Wythe Domaille A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Christopher J. Chang, Chair Professor Kenneth N. Raymond Professor Carolyn R. Bertozzi Professor Jhih-Wei Chu Spring 2011 Molecular Imaging Approaches to Understanding the Roles of Copper in Biology © 2011 by Dylan Wythe Domaille Abstract Molecular Imaging Approaches to Understanding the Roles of Copper in Biology by Dylan Wythe Domaille Doctor of Philosophy in Chemistry University of California, Berkeley Professor Christopher J. Chang, Chair Cells exert precise control over their cellular copper pools through a sophisticated array of uptake, trafficking, and storage mechanisms that effectively maintain a low concentration of thermodynamically free copper ions while maintaining excellent kinetic lability of cellular copper stores. In higher eukaryotes and humans, and particularly in specialized cell types associated with brain, heart, intestine, and liver tissue, the molecular specifics of how kinetically labile copper pools are regulated at the subcellular level and the consequences of copper misregulation in aging and disease remain insufficiently understood. Biochemical and genetic studies have established a broad understanding of how cells acquire, maintain, redirect and release copper ions, while also identifying key proteins involved in these activities.
    [Show full text]
  • Pentiptycene-Derived Fluorescence Turn-Off Polymer Chemosensor for Copper(II) Cation with High Selectivity and Sensitivity
    polymers Article Pentiptycene-Derived Fluorescence Turn-Off Polymer Chemosensor for Copper(II) Cation with High Selectivity and Sensitivity Anting Chen 1, Wei Wu 1, Megan E. A. Fegley 1, Sherryllene S. Pinnock 1, Jetty L. Duffy-Matzner 2, William E. Bernier 1 and Wayne E. Jones Jr. 1,* 1 Department of Chemistry, Binghamton University, State University of New York, Binghamton, NY 13902, USA; [email protected] (A.C.); [email protected] (W.W.); [email protected] (M.E.A.F.); [email protected] (S.S.P.); [email protected] (W.E.B.) 2 Department of Chemistry, Augustana University, Sioux Falls, SD 57197, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-607-777-2421 Academic Editor: Po-Chih Yang Received: 2 March 2017; Accepted: 21 March 2017; Published: 24 March 2017 Abstract: Fluorescent conjugated polymers (FCPs) have been explored for selective detection of metal cations with ultra-sensitivity in environmental and biological systems. Herein, a new FCP sensor, tmeda-PPpETE (poly[(pentiptycene ethynylene)-alt-(thienylene ethynylene)] with a N,N,N0-trimethylethylenediamino receptor), has been designed and synthesized via Sonogashira cross-coupling reaction with the goal of improving solid state polymer sensor development. The polymer was found to be emissive at lmax ~459 nm under UV radiation with a quantum yield of 0.119 at room temperature in THF solution. By incorporating diamino receptors and pentiptycene groups into the poly[(phenylene ethynylene)-(thiophene ethynylene)] (PPETE) backbone, the polymer showed an improved turn-off response towards copper(II) cation, with more than 99% quenching in fluorescence emission. It is capable of discriminating copper(II) cation from sixteen common cations, with a detection limit of 16.5 nM (1.04 ppb).
    [Show full text]
  • Zinc (II) and Aiegens: the “Clip Approach” for a Novel Fluorophore Family
    molecules Review Zinc (II) and AIEgens: The “Clip Approach” for a Novel Fluorophore Family. A Review Rosita Diana and Barbara Panunzi * Department of Agricultural Sciences, University of Naples Federico II, 80055 Portici, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-081-674-170 Abstract: Aggregation-induced emission (AIE) compounds display a photophysical phenomenon in which the aggregate state exhibits stronger emission than the isolated units. The common term of “AIEgens” was coined to describe compounds undergoing the AIE effect. Due to the recent interest in AIEgens, the search for novel hybrid organic–inorganic compounds with unique luminescence properties in the aggregate phase is a relevant goal. In this perspective, the abundant, inexpensive, and nontoxic d10 zinc cation offers unique opportunities for building AIE active fluorophores, sensing probes, and bioimaging tools. Considering the novelty of the topic, relevant examples collected in the last 5 years (2016–2021) through scientific production can be considered fully representative of the state-of-the-art. Starting from the simple phenomenological approach and considering different typological and chemical units and structures, we focused on zinc-based AIEgens offering synthetic novelty, research completeness, and relevant applications. A special section was devoted to Zn(II)- based AIEgens for living cell imaging as the novel technological frontier in biology and medicine. Keywords: AIE; zinc complex; fluorescence Citation: Diana, R.; Panunzi, B. Zinc (II) and AIEgens: The “Clip Approach” for a Novel Fluorophore 1. Introduction Family. A Review. Molecules 2021, 26, 1.1. Activation of the Fluorescence Channel in AIEgens 4176. https://doi.org/10.3390/ A luminescent material is a material able to emit light in the process of returning molecules26144176 from the electronic or vibrational excited state to the ground state after being excited by external energy.
    [Show full text]
  • Studying Zinc Homeostasis in Escherichia Coli Using Carbonic Anhydrase-Based Ratiometric Sensors
    STUDYING ZINC HOMEOSTASIS IN ESCHERICHIA COLI USING CARBONIC ANHYDRASE-BASED RATIOMETRIC SENSORS by Da Wang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2012 Doctoral Committee: Professor Carol A. Fierke, Chair Professor Raoul Kopelman Professor James E. Penner-Hahn Professor Joel A. Swanson © Da Wang 2012 DEDICATION To my parents. ii ACKNOWLEDGEMENTS There are many people I would like to thank through my graduate school. First, I would like to thank my advisor Dr. Carol Fierke for giving me great guidance and support through my graduate schooling. She has always been encouraging, understanding, helpful and supportive in both science and my personal life. I have learnt so much from her that will benefit me lifelong wherever I go. I would like to thank my committee, Prof. James Penner-Hahn, Prof. Raoul Kopelman, and Prof. Joel Swanson. I have benefited greatly from the helpful advice and insightful comments about my work over the past years. I truly appreciate the help. I would also like to thank my collaborators Dr. Richard Thompson and Dr. Bryan McCranor at the University of Maryland School Of Medicine. Dr. Thompson has been very instrumental in solving many of the practical issues of sensor development, and Dr. McCranor kindly provided assistance in sensor development for mammalian cell imaging. I appreciate their input that helped move the project forward. I would like to thank all my labmates for creating such a great and wonderful lab culture. It has been a delightful experience working with you.
    [Show full text]
  • Challenges and Opportunities for Small-Molecule Fluorescent Probes in Redox Biology Applications
    ANTIOXIDANTS & REDOX SIGNALING Volume 00, Number 00, 2018 ª Mary Ann Liebert, Inc. DOI: 10.1089/ars.2017.7491 FORUM REVIEW ARTICLE Challenges and Opportunities for Small-Molecule Fluorescent Probes in Redox Biology Applications Xiqian Jiang,1 Lingfei Wang,1 Shaina L. Carroll,1 Jianwei Chen,1 Meng C. Wang,2,3 and Jin Wang1,4,5 Abstract Significance: The concentrations of reactive oxygen/nitrogen species (ROS/RNS) are critical to various bio- chemical processes. Small-molecule fluorescent probes have been widely used to detect and/or quantify ROS/ RNS in many redox biology studies and serve as an important complementary to protein-based sensors with unique applications. Recent Advances: New sensing reactions have emerged in probe development, allowing more selective and quantitative detection of ROS/RNS, especially in live cells. Improvements have been made in sensing reactions, fluorophores, and bioavailability of probe molecules. Critical Issues: In this review, we will not only summarize redox-related small-molecule fluorescent probes but also lay out the challenges of designing probes to help redox biologists independently evaluate the quality of reported small-molecule fluorescent probes, especially in the chemistry literature. We specifically highlight the advantages of reversibility in sensing reactions and its applications in ratiometric probe design for quantitative measurements in living cells. In addition, we compare the advantages and disadvantages of small-molecule probes and protein-based probes. Future Directions: The low physiological relevant concentrations of most ROS/RNS call for new sensing reactions with better selectivity, kinetics, and reversibility; fluorophores with high quantum yield, wide wavelength coverage, and Stokes shifts; and structural design with good aqueous solubility, membrane permeability, low protein interference, and organelle specificity.
    [Show full text]
  • Visualization of Peroxynitrite-Induced Changes of Labile Zn[Superscript 2+] in the Endoplasmic Reticulum with Benzoresorufin-Based Fluorescent Probes
    Visualization of Peroxynitrite-Induced Changes of Labile Zn[superscript 2+] in the Endoplasmic Reticulum with Benzoresorufin-based Fluorescent Probes The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Lin, Wei, Daniela Buccella, and Stephen J. Lippard. “Visualization of Peroxynitrite-Induced Changes of Labile Zn2+ in the Endoplasmic Reticulum with Benzoresorufin-Based Fluorescent Probes.” Journal of the American Chemical Society 135, no. 36 (September 11, 2013): 13512–13520. As Published http://dx.doi.org/10.1021/ja4059487 Publisher American Chemical Society (ACS) Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/88575 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Visualization of Peroxynitrite-Induced Changes of Labile Zn2+ in the Endoplasmic Reticulum with Benzoresorufin- based Fluorescent Probes Wei Lin,‡ Daniela Buccella,‡,† and Stephen J. Lippard*,‡ ‡Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States †Department of Chemistry, New York University, New York, New York 10003, United States KEYWORDS: labile zinc, zinc fluorescent sensor, unfolded protein response, fluorescence imaging, benzoresorufin. ABSTRACT: Zn2+ plays essential roles in biology, and the homeostasis of Zn2+ is tightly regulated in all cells. Subcellular distribution and 2+ trafficking of labile Zn , and its interrelation with reactive nitrogen species, are poorly understood due to the scarcity of appropriate imaging tools. We report a new family of red-emitting fluorescent sensors for labile Zn2+, ZBR1-3, based on a benzoresorufin platform functionalized with dipicolylamine or picolylamine-derived metal binding groups.
    [Show full text]