ENHANCING THE FLUORESCENCE OF DNA-PROBES FOR MOLECULAR DIAGNOSTICS

Modern molecular diagnostics with polymerase chain reaction (PCR) is widely used as a basic tool for detection of various diseases and control of their course. Today the fluorescently labeled DNA-probes have the greatest popularity due to their high sensitivity and ease of use. Fluorescence intensity is the most important characteristic of fluorescently labeled DNA-probes. Increase of fluorescence intensity will enhance the sensitivity of analysis. Fluorescence intensity can be increased by multiple introduction of fluorophores based on special rigid linkers. Therefore the derivatives of 3,5-diaminobenzoic acid were used. In this paper we report on the synthesis of linkers based on 3,5-diaminobenzoic acid and their application in synthesis of the reagents with two fluorescein fluorophores and phosphoramidite or azide function for introduction of label into oligonucleotides. It is shown that obtained DNA-probes have almost twofold increase of fluorescence intensity and improved photophysical characteristics.

branching reagents, fluorescence, doubler, 3,5-diaminobenzoic acid, fluorescein, PCR, PCR-RT, fluorescence enhancement

1. Rebrikov D. V., Samatov G. A., Trofimov D. Iu., Semenov P. A., Savilova A. M., Kofiadi I. A., Abramov D. D., PTsR v real’nom vremeni [Real-time PCR], in Rebrikov D. V. (ed.), 2nd ed., BINOM. Laboratoriia znanii, Moscow, RU, 2009.

2. Wilson J.N., Teo Y.N., Kool E.T., “Efficient Quenching of Oligomeric Fluorophores on a DNA Backbone”, Journal of the American Chemical Society, 2007, vol. 129, pp. 15426–15427.

3. Hameau A., Fuchs S., Laurent R., Majoral J.-P., Caminade A.-M., “Synthesis of dye/fluorescent functionalized dendrons based on cyclotriphosphazene”, Beilstein journal of organic chemistry, 2011, vol. 7, no. 1, pp. 1577–1583.

4. Hahn C. D., Riener C. K., Gruber H. J., “Labeling of antibodies with Cy3-, Cy3. 5-, Cy5-, and Cy5. 5-monofunctional dyes at defined dye/protein ratios”, Single Molecules, 2001, vol. 2, no. 2, pp. 149–159.

5. Martin V. V., Alferiev I. S., Weis A. L., “Amplified fluorescent molecular probes based on 1, 3, 5, 7-tetrasubstituted adamantine”, Tetrahedron letters, 1999, vol. 40, no. 2, pp. 223–226.

6. Misra A., Mishra S., Misra K., “Synthesis and Fluorescence Studies of Multiple Labeled Oligonucleotides Containing Dansyl Fluorophore Covalently Attached at 2′-Terminus of Cytidine via Carbamate Linkage”, Bioconjugate Chemistry, 2004, vol. 15, pp. 638–646.

7. Tietze L., Eicher T., Preparativnaia organicheskaia khimiia. Reaktsii i sintezy v praktikume organicheskoi khimii i nauchno-issledovatel’skoi laboratorii [Preparative organic chemistry. Reactions and syntheses in the workshop of organic chemistry and research laboratory], Mir, Moscow, RU, 1999.

8. Guzaev A., Salo H., Azhayev A., Lönnberg H., “Novel Non-Nucleosidic Building Blocks for the Preparation of Multilabeled Oligonucleotides”, Bioconjugate Chemistry, 1996, vol. 7, pp. 240–248.