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The following paper appeared in Proceedings of the Fifty-Second Annual Meeting, Microscopy Society of America; Bailey, G. W., and Garratt-Reed, A. J., Eds.; San Francisco Press, San Francisco, CA, 1994, pp. 176-177.

Combined Fluorescent and Gold Nucleic Acid Probes


Richard D. Powell,* James F. Hainfeld,* Mair E. A. Churchill** and Andrew S. Belmont**

*Nanoprobes, Incorporated, Stony Brook, NY 11790
**Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801


Site-specific probes which incorporate both fluorophores and the 1.4 nm "Nanogold" gold particle 1 have been prepared (Fig. 1). Specimens labeled with these probes may be imaged by fluorescence microscopy, then examined at higher resolution in the electron microscope without an additional gold-labeling step: this allows better specimen preservation and unprecedented correlation between the two sets of complementary data. In addition, the covalent gold attachment procedure allows the preparation of electron microscopy probes using small molecules, which cannot be coupled to colloidal gold.

Two types of probes have been prepared. In the first, the Nanogold particle was covalently attached, via peripheral primary amine groups, to Hoescht-33258, a fluorescent dye which is specific for AT-rich regions of double-stranded DNA.2 Excess Hoescht-33258 was removed by membrane separation. The resulting reagent was tested in Chinese hamster ovary cells: results are shown in Figs. 2 and 3. Specific staining of the nucleus was observed both by fluorescence microscopy and in the light microscope, using silver enhancement (Danscher's procedure3) to visualize the gold-labeled regions.

In the second approach, a new label was prepared which comprises a small number of (estimated 2 to 4) fluorescein groups and the 1.4 nm Nanogold particle. This new label may be covalently linked to targeted biomolecules via a peripheral sulfo-N-hydroxysuccinimide or maleimide group.4 In a comparison with fluorescein isothiocyanate (FITC), fluorescence intensity relative to UV/visible absorbtion for the fluorescein moities conjugated to the Nanogold particle was found to be 0.83 (FITC = 1.00); previous attempts to prepare dual fluorescent and colloidal gold probes have resulted in substantial loss of fluorescence, possibly due to quenching by the colloidal gold particles.5

This label was conjugated to a small peptide from the C-terminal region of histone H1 protein, which is thought to recognize the minor groove in AT-rich regions of double-stranded DNA.6 Fluorescein/1.4 nm gold particles prepared with a peripheral primary amine were converted to the sulfo-N-hydroxysuccinimido- form by reaction with bis(sulfo-succinimidyl) suberate (BS3), separated from excess BS3 by gel filtration, then incubated with a 50-fold excess of the peptide at 4°C overnight. Labeled peptide was separated from unbound peptide by gel filtration. This probe selectively stained chromatin in Chinese hamster ovary cells, visualized by silver enhancement in the light microscope (Fig. 5). Electron microscope observation showed localization of the enlarged gold particles at sites containing DNA (nuclear chromatin) and RNA, as shown in Fig. 5. Additional confirming studies are planned using other fluorescent / gold labeled peptides and antibodies.7


References

  1. Hainfeld, J. F., and Furuya, F. R., J. Histochem. Cytochem. 40 (1992) 177.

  2. Churchill, M. E. A., and Travers, A. A., Trends Biochem. Sci., 16 (1991), 92.

  3. Danscher, G., Histochemistry, 71 (1981), 81.

  4. Hainfeld, J. F., Science, 236 (1987) 450.

  5. Goodman, S. L., Park, K., and Albrecht, R. M., in Colloidal Gold: Principles, Methods and Applications, Academic Press (1991) 370.

  6. Churchill, M. E. A., and Suzuki, M., EMBO J., 8 (1989) 4189.

  7. This work was supported by NIH grants GM 48328 (RDP, JFH) and GM 42516 (ASB).

[Figure 1] (9k)

[Figures 2-5: Micrographs] (100k)

Figure 1. (a) Nanogold coupled to Hoescht-33258; (b) Fluorescein/Nanogold labeled biomolecule (covalent attachment through primary amine group on biomolecule).

Figure 2. Chinese hamster ovary cells stained with Nanogold-Hoescht-33258: high magnification optical section after deconvolution. Mitotic chromosomes (lower left) show higher fluorescence intensity.

Figure 3. Intermediate-magnification light microscope image of cells stained with Nanogold-Hoescht 33258 after silver enhancement.

Figure 4. Low magnification light micrograph of chinese hamster ovary cells stained with fluorescein-Nanogold-H1 peptide, after silver enhancement.

Figure 5. Darkfield electron micrograph of cells stained with fluorescein-Nanogold-H1 peptide, after silver enhancement (bar = 0.5 micrometers).

Thanks to the San Francisco Press for allowing us to reproduce this online.


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