Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Aug;2(4):136-40.
doi: 10.1002/open.201300017. Epub 2013 Jun 21.

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

Carolin Holzhauser  1 , Sabrina Kracher  2 , Moritz M Rubner  1 , Wolfgang Schmucker  1 , Hans-Achim Wagenknecht  1 , Ralph Witzgall  2
Affiliations

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

Carolin Holzhauser et al. ChemistryOpen. 2013 Aug.
No abstract available

Keywords: cyanines; electron; fluorescence; microscopy; readout.

PubMed Disclaimer

Figures

Figure 1
Figure 1
A) Structure of fluorophore–DNA conjugates: perylene attached to the 5-position of 2′-desoxyuridine (PedU), cyanine indole quinolinium attached to the 2′-position of uridine (CyIQU) and thiazole orange as a DNA/RNA base substitution (TO). B) UV/Vis absorption. C) Fluorescence of DNA1–DNA4 and RNA5 at 2.5 μm in NaH2PO4/NaH2PO4 (50 mm, pH 7), NaCl (250 mm), 25 °C, λexc=426 nm (Pe), 490 nm (TO) or 495 nm (CyIQ).
Figure 2
Figure 2
Fluorescently labeled oligonucleotides are taken up by transiently transfected LLC-PK1 cells. LLC-PK1 cells were transfected with single-stranded oligonucleotide DNA2 (stock concentration: 100 μm) and Lipofectamine 2000. Fluorescent cytosolic structures (arrows) are shown A) 1 h, B) 2 h, C) 12 h and D) 24 h after the addition of the DNA/lipofectamine mixture (white bar represents 10 μm).
Figure 3
Figure 3
Fluorescent oligonucleotides are present in early endosomes and in lysosomes. LLC-PK1 cells were transfected with single-stranded oligonucleotide DNA2 (stock concentration: 100 μm) and Lipofectamine 2000. Two hours after the addition of the DNA/lipofectamine mixture, the cells were stained with primary antibodies against A) EEA1 and B) LAMP1, markers for early endosomes and lysosomes, respectively. The arrows point to EEA1- and LAMP1-positive structures containing fluorescent oligonucleotides (white bar represents 10 μm).
Figure 4
Figure 4
Electron microscopic detection of internalized oligonucleotides. LLC-PK1 cells were transfected with single-stranded DNA oligonucleotide DNA2 (stock concentration: 100 μm) and Lipofectamine 2000. Fluorescent cells were photooxidized in the presence of DAB. In some cells, a labeling of the plasma membrane could be detected (A,B). A membrane-lined cytosolic structure contains electron-dense precipitates and additional small vesicles, and therefore can be identified as a late endosome (C). Few nuclei contained electron-dense structures that could be clearly differentiated from heterochromatin (D, E). The chromatin staining in D) seems to be an artifact. White bar represents 1000 nm (A,B,D) or 250 nm (C,E).
See this image and copyright information in PMC

References

    1. Kobayashi H, Ogawa M, Alford R, Choydke PL, Urano Y. Chem. Rev. 2010;110:2620–2640. - PMC - PubMed
    1. Berezin MY, Achilefu S. Chem. Rev. 2010;110:2641–2684. - PMC - PubMed
    1. Sinkeldam RW, Greco NJ, Tor Y. Chem. Rev. 2010;110:2579–2619. - PMC - PubMed
    1. Lavis LD, Raines RT. ACS Chem. Biol. 2008;3:142–155. - PMC - PubMed
    1. Waggoner A. Curr. Opin. Chem. Biol. 2006;10:62–66. - PubMed