Photochemically Active Fluorophore–DNA/RNA Conjugates for Cellular Imaging of Nucleic Acids by Readout in Electron Microscopy

Imaging is a key research goal for the understanding of biological processes by proteins or nucleic acids inside the living cell. By using classical confocal fluorescence microscopy it is possible to track those biomolecules that are labeled covalently with a suitable fluorophore or fluorescent protein. Fluorescence microscopy as a noninvasive and nondestructive method (if the applied fluorophores are sufficiently photostable) offers the advantage to follow biological processes in real time over hours or even days. Over the last two decades, advanced fluorescence spectroscopy methods, such as stimulated emission depletion (STED), photoactivated localization microscopy (PALM) and others, have revitalized light-based microscopy for cell biology. Additionally, cell images obtained by electron microscopy allow the characterization of subcellular structures. The most complete picture would be drawn from imaging, if dynamic fluorescence and static electron microscopy were combined and correlated by applying a single label that gives readout in both types of microscopy. So far, this has been achieved to a certain extent for observing and localizing lipids and proteins inside cells but not for nucleic acids. The idea to use fluorescent chromophores not only for fluorescence microscopy but additionally as chromogenes to stain electron microscopy images has been published for the first time in 1982 to image neurons. Since then, a variety of examples were presented mainly for proteins. The most successful technique is to use the fluorophores or fluorescent proteins themselves in order to photooxidize 3,3’-diaminobenzidine (DAB) which initiates polymerization. 15–17] The resulting polymer can be stained by heavy atom salts, such as OsO4, and finally be visualized in histological sections by electron microscopy. Proteins were covalently and noncovalently labeled with biarsenical fluorophores, fluorescent proteins, 4’,6-diamidino-2-phenylindole (DAPI), eosin, lucifer yellow propidium iodide or quantum dots to get specific readout not only in confocal fluorescence spectroscopy but also in electron microscopy. To our knowledge, however, the methodology by photoinduced DAB polymerization has never been transferred to image nucleic acids. There is only one example of staining nucleic acids in electron micrographs, which, however, uses a completely different technique. The goal of this work was to identify labels that enable a readout in both types of microscopy. Herein, we present the photochemical evaluation of three different fluorophores covalently attached to short DNA and RNA strands and their ability to image nucleic acids inside cells both by confocal microscopy and by well-resolved snapshots taken by electron microscopy. We chose representatively the delivery process of nucleic acids into cells to test the dual readout of our synthetic DNA– and RNA–conjugates in both imaging methods. It is important to mention here that we recently have shown that similarly modified siRNA exhibits only slightly reduced functionality in comparison to non-modified RNA. Although the mechanism of photochemically induced polymerization of DAB is not completely understood, recent studies with eosin and other chromophores revealed that singlet oxygen (O2) and oxygen radicals (such as the superoxide anion O2C ) may play a key role as initiators of photopolymerization. These reactive oxygen species are formed in situ upon excitation of chromophores. Direct photooxidation has been identified as an alternative pathway, since DAB is a good electron donor (Eox=0.335 V vs. Ag /AgCl). Using a conversion constant of ca. +0.55 V from the Ag/AgCl electrode to the normal hydrogen electrode (NHE), DAB exhibits an oxidation potential of approximately 0.9 V vs. NHE. Hence, we identified for our studies three fluorescent chromophores with suitable redox potentials and excitation energies that are sufficient for direct photooxidation of DAB. These are (1) perylene (Pe) conjugated to the 5-position of 2’-desoxyuridine in DNA1, (2) cyanine indole quinolinium (CyIQ) attached to the 2’-position of uridine in DNA2 and DNA3, and (3) thiazole orange (TO) as a base substitution in DNA4 and RNA5 (Figure 1; Table 1). The latter chromophore (TO), a fluorophore well known from noncovalent staining agents like TOTO, shows a reduction potential of 1.0 V (vs. NHE). According to the Rehm–Weller equation DG=Eox Ered E00 (without the Coulomb contribution) and E00=2.4 eV for TO, the [a] Dr. C. Holzhauser, Dr. M. M. Rubner, Dr. W. Schmucker, Prof. Dr. H.-A. Wagenknecht Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT) Fritz-Haber-Weg 6, 76131 Karlsruhe (Germany) E-mail : [email protected] [b] Dr. S. Kracher, Prof. Dr. R. Witzgall Institute of Molecular and Cellular Anatomy, University of Regensburg Universit tsstr. 31, 93053 Regensburg (Germany) E-mail : [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/open.201300017. 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.