DOCSLIB.ORG

Orientation of Cyanine Fluorophores Terminally Attached to DNA Via Long, Flexible Tethers

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Orientation of Cyanine Fluorophores Terminally Attached to DNA Via Long, Flexible Tethers 1148 Biophysical Journal Volume 101 September 2011 1148–1154 Orientation of Cyanine Fluorophores Terminally Attached to DNA via Long, Flexible Tethers Jonathan Ouellet, Stephanie Schorr, Asif Iqbal, Timothy J. Wilson, and David M. J. Lilley* Cancer Research UK Nucleic Acid Structure Research Group, The University of Dundee, Dundee, United Kingdom ABSTRACT Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 50-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 50-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocya- nine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindo- carbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by ~60 for the long- tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species. INTRODUCTION Fluorescence resonance energy transfer (FRET) is exten- J(l) is the spectral overlap between donor emission and sively used to measure or compare distances in biological acceptor absorption, given by macromolecules (1), especially nucleic acids (2–5). It RN 4 provides distance information in a size range suitable for 4DðlÞεAðlÞl dl biological macromolecules, and because of the sensitivity JðlÞ¼ 0 (3) RN of fluorescence, it can be performed on single molecules 4DðlÞdl (6). However, the interpretation of FRET data is potentially 0 complicated by effects arising from the orientation of the fluorophores used. where 4D(l) is the spectral shape of donor emission and FRET arises from the dipolar coupling between the εA(l) the extinction coefficient for acceptor absorption transition moment vectors of fluorophores, a function of (MÀ1 cmÀ1). The value k2 describes the relative orientation distance and orientation. The efficiency of FRET (EFRET) of the fluorophores. It is given by as a function of fluorophore separation (R) is given by 2 2 Fo¨rster (7)as k ¼ðcos QT À 3  cos QD  cos QAÞ (4) 1 E ¼ (1) FRET 6 where the angles QT, QD, and QA describe the relative 1 þ R= orientation of the donor and acceptor transition dipole R0 moments (Fig. 1 A). Uncertainty in the value of k2 (8–10) provides the largest potential difficulty in the extraction of 2 where R0 is the distance at which energy transfer is 50% distance information from EFRET. The value k can take efficient. R0 depends upon the spectroscopic properties of values between 0 and 4, or between 0 and 1 if the transition the fluorophores and the medium, given by moments are coaxial and constrained to parallel planes. If 0:529  k2  f  JðlÞ the fluorophores undergo isotropic reorientation within the R6 ¼ D (2) k2 ¼ 0 N  n4 excited state lifetime of the donor, then 2/3 (9), thus simplifying the extraction of distance information. In many studies this is assumed to apply, explicitly or other- where the units of R and the wavelength l are cm. The 0 wise, but this may not necessarily be warranted. There has value f is the quantum yield of the donor, N is the Avoga- D been renewed interest in this problem recently (11–13). dro number, n is the index of refraction of the medium, and The cyanine fluorophores Cy3 and Cy5 are extensively used as a FRET donor acceptor pair attached to nucleic Submitted June 1, 2011, and accepted for publication July 11, 2011. acids, particularly in single-molecule studies (14). We have shown that the fluorophores Cy3 and Cy5 stack on *Correspondence: [email protected] 0 Editor: David P. Millar. the ends of DNA double helices when attached to the 5 Ó 2011 by the Biophysical Society 0006-3495/11/09/1148/7 $2.00 doi: 10.1016/j.bpj.2011.07.007 Cyanine Fluorophore Orientation on DNA 1149 2 A that an assumption of k ¼ 2/3 is not justified. This was demonstrated by making a series of DNA and DNA-RNA duplexes labeled at their 50-termini with Cy3 and Cy5 (12). We observed that the efficiency of energy transfer from Cy3 donor to Cy5 acceptor as a function of the length of the helix was significantly modulated in a periodic manner, with maxima and minima occurring twice per helix rotation. The profiles of EFRET as a function of helix length could be well simulated assuming standard B- and A-form helical geometries and terminally stacked fluorophores that can B undergo a significant degree of lateral mobility about a mean position deduced from NMR studies (12). Because the calculated transition moments for the p to p* transitions lie in the plane of the indole rings close to the poly-methine linker (16), the fluorophore positions are close to satisfying the conditions for a variation of 0 % k2 % 1, and the dipoles should be perpendicular twice per helical period (i.e., k2 ¼ 0) (Fig. 1 C). In principle, the orientational effects could be greater if the fluorophores planes are not parallel, for example when terminally attached to a helix that is bent by some discontinuity such as a junction or bulge. In that case, k2 could vary over its full range from 0 to 4. In our previous studies, the cyanine fluorophores were 0 linked to 5 -termini via a C3 linker. This is the same number of carbon atoms that exist when linked via a ribose ring, and C it is possible that this constrains the fluorophores to stack on the end of the helix in the manner of an additional nucleo- tide base. A commonly-used alternative approach is to con- jugate the fluorophores as N-hydroxysuccinimide esters to 0 a primary amine that is linked to the 5 -terminus by a C6 tether, generating a total tether length of 13 atoms. The structures created by use of the C3-linked phosphoramidite and conjugation using the C6 linker are compared in Fig. 2. In principle, the longer linker could provide much greater FIGURE 1 Orientation of the transition moments of cyanine fluoro- flexibility than the C3 tether. The structure of the fluoro- phores attached to DNA. (A) The orientation parameter k2. The transition phores used in the two methods also differs. The Cy3 and dipole vectors for the coupled donor and acceptor fluorophore are indicated 0 Cy5 N-hydroxysuccinimide esters carry sulfonyl groups at (arrows), labeled D and A. Vector A is generated by the in-plane translation 0 of vector A to share its origin with vector D. The definition of k2, given in the 5 and 5 positions of the indole rings. The presence of Eq. 4, is based upon the angles shown. (B) Parallel-eye stereoscopic view these negative charges could affect the stacking of the fluo- of a model of a duplex with Cy3 and Cy5 fluorophores attached to the rophores (17). The longer linker and altered structure of the 0 5 -termini via C3 linkers. This was generated using our NMR structures fluorophore might conceivably remove a constraint that of Cy3 and Cy5 attached to duplex DNA. The fluorophores lie in approxi- requires it to stack on the terminal basepair. On the other mately parallel planes, and thus the angular relationship between them (and thus their transition moments) will depend on the length of the DNA helix, hand, perhaps the tendency to stack is intrinsic to the and its helical periodicity. (C) Schematic to illustrate the principle of the cyanine fluorophores in general, and will occur irrespective experiment in which a series of DNA duplexes terminally labeled with of the nature of the linkage and substitution in the indole Cy3 and Cy5 is used. The cyanine fluorophores are represented as flat disks, ring. We have therefore set out to answer this question, with the direction of the transition moments indicated (arrows). As the helix which is of great practical significance for the interpretation length is increased there is a rotation of the relative angle between the two k2 of FRET experiments using these fluorophores. transition dipoles. The term takes the value 1 when they are parallel, and 0 0 when perpendicular. We have used a 10–24 bp duplex series with 5 sulfoindo- carbocyanine 3 and 5 attached via long tethers to examine whether the orientation observed with the shorter tether is termini via three-methylene carbon (C3) tethers generated maintained. We find significant modulation of EFRET with by coupling as phosphoramidites (15,16)(Fig. 1 B). This helix length measured in single-molecule experiments, generates a significant orientation of the fluorophores, so leading to the conclusion that the orientation of the cyanine Biophysical Journal 101(5) 1148–1154 1150 Ouellet et al. A Phospholipid encapsulation A 100:1 molar ratio of unmodified/biotinylated phospholipids was prepared by evaporating a number of aliquots of a mixture of 2.5 mg L-a-phosphatidylcholine and 35 mg 1,2-dipalmitoyl-sn-glycero-3-phos- phoethanolamine-N-(cap biotinyl) (Avanti Polar Lipids, Alabaster, AL) from chloroform in a stream of argon.
Load more

Recommended publications
  • The Green Fluorescent Protein
    P1: rpk/plb P2: rpk April 30, 1998 11:6 Annual Reviews AR057-17 Annu. Rev. Biochem. 1998. 67:509–44 Copyright c 1998 by Annual Reviews. All rights reserved THE GREEN FLUORESCENT PROTEIN Roger Y. Tsien Howard Hughes Medical Institute; University of California, San Diego; La Jolla, CA 92093-0647 KEY WORDS: Aequorea, mutants, chromophore, bioluminescence, GFP ABSTRACT In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrin- sically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the rela- tion between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories. CONTENTS NATURAL AND SCIENTIFIC HISTORY OF GFP .................................510 Discovery and Major Milestones .............................................510 Occurrence, Relation to Bioluminescence, and Comparison with Other Fluorescent Proteins .....................................511 PRIMARY, SECONDARY, TERTIARY, AND QUATERNARY STRUCTURE ...........512 Primary Sequence from
    [Show full text]
  • Some New Nitrogen Bridgehead Heterocyclic Cyanine Dyes
    Some New Nitrogen Bridgehead Heterocyclic Cyanine Dyes A. I. M. KORAIEM, H. A. SHINDY, and R. M. ABU EL-HAMD Department of Chemistry, Aswan Faculty of Science, Aswan, Egypt Received 21 July 1998 Accepted for publication 30 December 1999 New monomethine, trimethine, and azomethine cyanine dyes of pyrazolo^o'iSjôJÍpyrazinio/l^- oxa£Ínio)[2,3,4-ij']quinolin-ll-ium bromides were prepared. These cyanines were identified by ele­ mental and spectral analyses. The visible absorption spectra of some selected dyes were investigated in single and mixed solvents, and also in aqueous buffer solutions. The spectral shifts are discussed in relation to molecular structure and in terms of medium effects. Molecular complex formation with DMF was verified through mixed-solvent studies. As an extension to our earlier work on the syn­ Extra quaternization of the former intermediates thesis and studies of cyanine dyes [1—3], some new using an excess amount of ethyl iodide under con­ nitrogen bridgehead heterocyclic cyanine dye moi­ trolled conditions gave 9-ethyl-8-R-10-methylpyrazo- eties have been synthesized in view of the applica­ lo[4^5^5,6](pyrazinio/l,4-oxazinio) [2,3,4- zj']quinolin- bility of such compounds in production of photother- ll(9)-ium bromides iodides (IVa—IVe). Further re­ mographic imaging materials [4], electrophotographic action of the latter compounds with iV-methylpyridi- lithographic printing material for semiconductor laser nium, -quinolinium, resp. -isoquinolinium iodide in­ exposure [5], and as indicator and pH sensors [6], or volving active hydrogen atom in the position 11 or as antitumour agents [7]. 8 under piperidine catalysis gave the correspond­ Within these respects, pyrazolo[4',5':5,6](pyrazi- ing unsymmetrical bromides iodides of 9-ethyl-8-R- nio/1,4-oxazinio) [2,3,4- i,j\ quinolin-11-ium bromides pyrazolo[4^5^5,6](pyrazinio/l,4-oxazinio) [2,3,4- i,j]- Ilia—IIIc were used as key intermediates for the dye quinolin-ll-ium-10[4(l)]-monomethine cyanine dyes synthesis.
    [Show full text]
  • Microenvironment-Triggered Dual-Activation of a Photosensitizer
    www.nature.com/scientificreports OPEN Microenvironment‑triggered dual‑activation of a photosensitizer‑ fuorophore conjugate for tumor specifc imaging and photodynamic therapy Chang Wang1, Shengdan Wang1, Yuan Wang1, Honghai Wu1, Kun Bao2, Rong Sheng1* & Xin Li1* Photodynamic therapy is attracting increasing attention, but how to increase its tumor‑specifcity remains a daunting challenge. Herein we report a theranostic probe (azo‑pDT) that integrates pyropheophorbide α as a photosensitizer and a NIR fuorophore for tumor imaging. The two functionalities are linked with a hypoxic‑sensitive azo group. Under normal conditions, both the phototoxicity of the photosensitizer and the fuorescence of the fuorophore are inhibited. While under hypoxic condition, the reductive cleavage of the azo group will restore both functions, leading to tumor specifc fuorescence imaging and phototoxicity. The results showed that azo‑PDT selectively images BEL‑7402 cells under hypoxia, and simultaneously inhibits BEL‑7402 cell proliferation after near‑infrared irradiation under hypoxia, while little efect on BEL‑7402 cell viability was observed under normoxia. These results confrm the feasibility of our design strategy to improve the tumor‑ targeting ability of photodynamic therapy, and presents azo‑pDT probe as a promising dual functional agent. Cancer is one of the most common causes of death, and more and more therapeutic strategies against this fatal disease have emerged in the past few decades. Among these strategies, photodynamic therapy has attracted much attention1. Tis therapy is based on singlet oxygen produced by photosensitizers under the irradiation with light of a specifc wavelength to damage tumor tissues (Fig. 1a). Since the photo-damaging efect is induced by the interaction between a photosensitizer and light, tumor-specifc therapy may be realized by focusing the light to the tumor site.
    [Show full text]
  • Cyanine Dye–Nucleic Acid Interactions
    Top Heterocycl Chem (2008) 14: 11–29 DOI 10.1007/7081_2007_109 © Springer-Verlag Berlin Heidelberg Published online: 23 February 2008 Cyanine Dye–Nucleic Acid Interactions Bruce A. Armitage Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA [email protected] 1Introduction................................... 12 2 Symmetrical Cyanines ............................. 14 2.1 NoncovalentBinding.............................. 14 2.2 CovalentBinding................................ 16 3 Unsymmetrical Cyanines ............................ 17 3.1 NoncovalentBinding.............................. 17 3.1.1InsightsintoPhotophysics........................... 17 3.1.2NewUnsymmetricalDyes............................ 18 3.1.3Applications................................... 21 3.2 CovalentBinding................................ 23 3.2.1DNA-ConjugatedDyes............................. 24 3.2.2PNA-ConjugatedDyes.............................. 25 3.2.3Peptide-andProtein-ConjugatedDyes.................... 26 4Conclusions................................... 27 References ....................................... 28 Abstract Cyanine dyes are widely used in biotechnology due to their ability to form fluor- escent complexes with nucleic acids. This chapter describes how the structure of the dye determines the mode in which it binds to nucleic acids as well as the fluorescence prop- erties of the resulting complexes. Related dyes, such as hemicyanines and styryl dyes, are briefly described as well. In addition, covalent conjugates
    [Show full text]
  • Fluorescent Protein-Based Tools for Neuroscience
    !1 !2 Fluorescent protein-based tools Outline for neuroscience An animatd primer on biosensor development Fluorescent proteins (FPs) Robert E. Campbell Department of Chemistry Other fluorophore technologies Single FP-based biosensors Imaging Structure & Function in the Nervous System Cold Spring Harbor, July 31, 2019. Lots of structural Lots of structural information information & Transmitted light Fluorescence microscopy of fluorescent color microscopy of live cells live cells No molecular provides molecular information information (more colors = more information) !5 !6 Fluorescence microscopy requires fluorophores Non-natural fluorophores for protein labelling Trends Bioch. Sci., 1984, 9, 88-91. O O O N O N 495 nm 519 nm 557 nm 576 nm - - CO2 CO2 S O O O N O N C N N C S ϕ = quantum yield - - ε = extinction coefficient CO2 CO2 ϕ Brightness ~ * ε S C i.e., for fluorescein N Proteins of interest ϕ = 0.92 N Fluorescein Tetramethylrhodamine ε = 73,000 M-1cm-1 C S (FITC) (TRITC) A non-natural fluorophore must be chemically linked to Non-natural fluorophores made by chemical synthesis a protein of interest… !7 !8 Getting non-natural fluorophores into a cell Some sea creatures make natural fluorophores Trends Bioch. Sci., 1984, 9, 88-91. O O O N O N Bioluminescent - Fluorescent CO2 CO -- S 2 S C N NHN C H S S Chemically labeled proteins of interest Microinjection with micropipet O O O N O N Fluorescent - CO2 CO - S 2 N NH H S …and then manually injected into a cell Some natural fluorophores are genetically encoded proteins http://www.luminescentlabs.org/and can be transplanted into cells as DNA! 228 OSAMU SHIMOMURA, FRANK H.
    [Show full text]
  • Fluorophore Referenceguide
    Fluorophore Reference Guide Fluorophore Excitation and Emission Data Laser Lines Broad UV Excitation Excitation Maxima Emission Maxima Emission Filters 290-365 nm LP = Long pass filter DF = Band pass filter Excel. ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ DAPI: 359 nm ____ SP = Short pass filter Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ GFP (Green Fluorescent Protein): 395 nm ____ 400 nm Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Coumarin: 402 nm ____ 425 nm Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ AttoPhos: 440 nm ____ ____ 443 nm: Coumarin 450 nm Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Acridine Orange: 460/500 nm ____ ____ 461 nm: DAPI Good __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ R-phycoerythrin: 480/565 nm ____ Excel.
    [Show full text]
  • Synthesis of Near-Infrared Heptamethine Cyanine Dyes
    Georgia State University ScholarWorks @ Georgia State University Chemistry Theses Department of Chemistry 4-26-2010 Synthesis of Near-Infrared Heptamethine Cyanine Dyes Jamie Loretta Gragg Georgia State University, [email protected] Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_theses Recommended Citation Gragg, Jamie Loretta, "Synthesis of Near-Infrared Heptamethine Cyanine Dyes." Thesis, Georgia State University, 2010. https://scholarworks.gsu.edu/chemistry_theses/28 This Thesis is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Theses by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. SYNTHESIS OF NEAR-INFRARED HEPTAMETHINE CYANINE DYES by JAMIE L. GRAGG Under the Direction of Dr. Maged M. Henary ABSTRACT Carbocyanine dyes are organic compounds containing chains of conjugated methine groups with electron-donating and electron-withdrawing substituents at the terminal 1 2 + - heterocycles of the general formula [R -(CH)n-R ] X . The synthetic methodology and optical properties of carbocyanines will be discussed. This thesis consists of two parts: (A) synthesis and optical properties of novel carbocyanine dyes substituted with various amines and the synthesis of unsymmetrical carbocyanine dyes containing monofunctional groups for bioconjugation. (B) synthesis of heptamethine carbocyanine dyes to be used for image-guided surgery. ii In part A, the synthesis of carbocyanine dyes functionalized with various amines and studies of their optical properties with respect to absorbance, fluorescence, quantum yield and extinction coefficient will be presented. These property studies will aid in designing efficient dyes for future biomedical applications.
    [Show full text]
  • Two-Photon Excited Photoconversion of Cyanine-Based Dyes
    Two-photon excited photoconversion of cyanine-based dyes The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Kwok, Sheldon J. J. et al. “Two-Photon Excited Photoconversion of Cyanine-Based Dyes.” Scientific Reports 6 (2016): 23866. © 2016 Macmillan Publishers Limited As Published http://dx.doi.org/10.1038/srep23866 Publisher Nature Publishing Group Version Final published version Citable link http://hdl.handle.net/1721.1/108348 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/4.0/ www.nature.com/scientificreports OPEN Two-photon excited photoconversion of cyanine-based dyes Sheldon J. J. Kwok1,2,*, Myunghwan Choi1,3,*, Brijesh Bhayana1, Xueli Zhang4,5, Chongzhao Ran4 & Seok-Hyun Yun1,2 Received: 14 October 2015 The advent of phototransformable fluorescent proteins has led to significant advances in optical Accepted: 15 March 2016 imaging, including the unambiguous tracking of cells over large spatiotemporal scales. However, these Published: 31 March 2016 proteins typically require activating light in the UV-blue spectrum, which limits their in vivo applicability due to poor light penetration and associated phototoxicity on cells and tissue. We report that cyanine- based, organic dyes can be efficiently photoconverted by nonlinear excitation at the near infrared (NIR) window. Photoconversion likely involves singlet-oxygen mediated photochemical cleavage, yielding blue-shifted fluorescent products. Using SYTO62, a biocompatible and cell-permeable dye, we demonstrate photoconversion in a variety of cell lines, including depth-resolved labeling of cells in 3D culture. Two-photon photoconversion of cyanine-based dyes offer several advantages over existing photoconvertible proteins, including use of minimally toxic NIR light, labeling without need for genetic intervention, rapid kinetics, remote subsurface targeting, and long persistence of photoconverted signal.
    [Show full text]
  • Water-Soluble Pyrrolopyrrole Cyanine (Ppcy) NIR Fluorophores† Cite This: Chem
    Erschienen in: Chemical Communications ; 2014, 50. - S. 4755-4758 ChemComm View Article Online COMMUNICATION View Journal | View Issue Water-soluble pyrrolopyrrole cyanine (PPCy) NIR fluorophores† Cite this: Chem. Commun., 2014, 50, 4755 Simon Wiktorowski, Christelle Rosazza, Martin J. Winterhalder, Ewald Daltrozzo Received 7th February 2014, and Andreas Zumbusch* Accepted 21st March 2014 DOI: 10.1039/c4cc01014k www.rsc.org/chemcomm Water-soluble derivatives of pyrrolopyrrole cyanines (PPCys) have been dyes, BODIPYs or others.7 Notable are also advances in other fields, synthesized by a post-synthetic modification route. In highly polar like the engineering of GFP-related fluorescing proteins or quantum media, these dyes are excellent NIR fluorophores. Labeling experiments dots, which have resulted in the synthesis of novel systems with NIR show how these novel dyes are internalized into mammalian cells. emission.8 To date, however, only a few water-soluble dyes with strong NIR absorptions and emissions have been known. Apart from the Near-infrared (NIR) light absorbing and emitting compounds have general scarcity of NIR absorbing molecules, the main reason for this attracted a lot of interest since the 1990’s.1 Initially, this was motivated is that NIR absorption is commonly observed in extended p-systems by their use in optical data storage or as laser dyes. Recently, however, which most often are hydrophobic. The incorporation of hydrophilic new applications of NIR dyes have emerged, which has led to a surge of functionalities into
    [Show full text]
  • Chiral Indole Intermediates and Their Fluorescent
    (19) TZZ_ _T (11) EP 1 525 265 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C09B 23/02 (2006.01) G01N 33/58 (2006.01) 20.07.2011 Bulletin 2011/29 G01N 33/533 (2006.01) C12Q 1/00 (2006.01) C07D 491/22 (2006.01) C07D 403/06 (2006.01) (21) Application number: 03808367.1 (86) International application number: (22) Date of filing: 09.05.2003 PCT/US2003/014632 (87) International publication number: WO 2004/039894 (13.05.2004 Gazette 2004/20) (54) CHIRAL INDOLE INTERMEDIATES AND THEIR FLUORESCENT CYANINE DYES CONTAINING FUNCTIONAL GROUPS CHIRALE INDOLE ALS ZWISCHENPRODUKTE UND DARAUS HERGESTELLTE CYANINFLUORESZENZFARBSTOFFE MIT FUNKTIONELLEN GRUPPEN INTERMEDIAIRES CHIRAUX D’INDOLE ET LEURS COLORANTS DE CYANINE FLUORESCENTS CONTENANT DES GROUPES FONCTIONNELS (84) Designated Contracting States: • WEST, Richard Martin, AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Amersham Biosciences UK Ltd. HU IE IT LI LU MC NL PT RO SE SI SK TR Buckinghamshire HP7 9NA (GB) (30) Priority: 10.05.2002 US 379107 P (74) Representative: Franks, Barry Gerard et al GE Healthcare Limited (43) Date of publication of application: Amersham Place 27.04.2005 Bulletin 2005/17 Little Chalfont Buckinghamshire HP7 9NA (GB) (60) Divisional application: 10178959.2 / 2 258 776 (56) References cited: WO-A-02/26891 US-A- 5 268 486 (73) Proprietors: US-A- 6 133 445 US-A- 6 133 445 • CARNEGIE MELLON UNIVERSITY US-B1- 6 180 087 Pittsburgh, Pennsylvania 15213 (US) • GE Healthcare Limited Remarks: Little Chalfont Thefilecontainstechnicalinformationsubmittedafter Buckinghamshire HP7 9NA (GB) the application was filed and not included in this specification (72) Inventors: • MUJUMDAR, Ratnaker, B.
    [Show full text]
  • Chemical Probes to Visualize Bacterial Cell Structure and Physiology
    molecules Review From Differential Stains to Next Generation Physiology: Chemical Probes to Visualize Bacterial Cell Structure and Physiology Jonathan Hira 1, Md. Jalal Uddin 1 , Marius M. Haugland 2 and Christian S. Lentz 1,* 1 Research Group for Host-Microbe Interactions, Department of Medical Biology and Centre for New Antibacterial Strategies (CANS), UiT—The Arctic University of Norway, 9019 Tromsø, Norway; [email protected] (J.H.); [email protected] (M.J.U.) 2 Department of Chemistry and Centre for New Antibacterial Strategies (CANS), UiT—The Arctic University of Norway, 9019 Tromsø, Norway; [email protected] * Correspondence: [email protected] Academic Editor: Steven Verhelst Received: 30 September 2020; Accepted: 23 October 2020; Published: 26 October 2020 Abstract: Chemical probes have been instrumental in microbiology since its birth as a discipline in the 19th century when chemical dyes were used to visualize structural features of bacterial cells for the first time. In this review article we will illustrate the evolving design of chemical probes in modern chemical biology and their diverse applications in bacterial imaging and phenotypic analysis. We will introduce and discuss a variety of different probe types including fluorogenic substrates and activity-based probes that visualize metabolic and specific enzyme activities, metabolic labeling strategies to visualize structural features of bacterial cells, antibiotic-based probes as well as fluorescent conjugates to probe biomolecular uptake pathways. Keywords: activity-based probe; antibiotic conjugate; bacterial imaging; bacterial uptake; fluorogenic substrate; metabolic labeling; phenotypic heterogeneity 1. Introduction—From 19th Century Microbiology to Modern Day Chemical Biology If chemical biology can be defined as the ‘interrogation of biological systems with chemical approaches’ [1], we must acknowledge some of the first microbiologists as chemical biologists.
    [Show full text]
  • Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome
    Near-infrared genetically encoded positive calcium indicator based on GAF-FP bacterial phytochrome The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Subach,Oksana M., Natalia V. Barykina, Konstantin V. Anokhin, Kiryl D. Piatkevich, and Fedor V. Subach, "Near-infrared genetically encoded positive calcium indicator based on GAF-FP bacterial phytochrome." International Journal of Molecular Sciences 20, 14 (July 2019): no. 3488 doi 10.3390/ijms20143488 ©2019 Author(s) As Published 10.3390/ijms20143488 Publisher Multidisciplinary Digital Publishing Institute Version Final published version Citable link https://hdl.handle.net/1721.1/125333 Terms of Use Creative Commons Attribution Detailed Terms https://creativecommons.org/licenses/by/4.0/ International Journal of Molecular Sciences Article Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome 1, 2, 2,3 Oksana M. Subach y , Natalia V. Barykina y , Konstantin V. Anokhin , Kiryl D. Piatkevich 4 and Fedor V. Subach 1,* 1 National Research Center “Kurchatov Institute”, Moscow 123182, Russia 2 P.K. Anokhin Institute of Normal Physiology, Moscow 125315, Russia 3 Lomonosov Moscow State University, Moscow 119991, Russia 4 MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA * Correspondence: [email protected]; Tel.: +07-968-962-7083 These authors contributed equally to this work. y Received: 31 May 2019; Accepted: 15 July 2019; Published: 16 July 2019 Abstract: A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum.
    [Show full text]