Biopolym. Cell. 2007; 23(2):137-142.
Bioorganic Chemistry
Synthesis of 3'- and 3',5'-modified oligonucleotides on functionalized silica Silochrom-2
1Dubey I. Ya., 1Dubey L. V., 2Fedoryak D. M.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine
    1, Murmans'ka Str., Kyiv, Ukraine, 02094


The preparation of 3'-mono- and 3', 5' -bis-labeled oligonucleotides on a new fluorescein-functionalized polymer support based on Silochrom-2 microspherical silica is described. Oligonucleotide (dT)15 containing a dye residue at 3'-end was synthesized on this support by solid-phase phosphoramidite method. Post-synthetic 5'-modification of 3'-conjugate allowed obtaining double dye-labeled oligomer. In the future it will be used for the synthesis of fluorescent probe for nucleic acids detection.
Keywords: oligonucleotide conjugates, fluorescent labels, solidphase synthesis, functionalization


[1] Goodchild J. Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconjug Chem. 1990;1(3):165-87.
[2] Beaucage SL, Iyer RP. The Functionalization of Oligonucleotides Via Phosphoramidite Derivatives. Tetrahedron. 1993;49(10):1925–63.
[3] Protocols for oligonucleotide conjugates: synthesis and analytical techniques. Methods in molecular biology. Ed. S. Agrawal. Totowa: Humana press, 1993. Vol. 26. 390 p.
[4] Korshun VA, Berlin YuA. Introduction of nonradioactive reporter groups into synthetic oligonucleotides and their detection. Bioorg Khim. 1994; 20(6):565-616.
[5] Synthesis of modified oligonucleotides and conjugates. Current Protocols in Nucleic Acid Chemistry. Eds S. L Beaucage et al. New York: John Wiley & Sons, 2003: 4.0.1-4.9.28.
[6] Reese CB. Oligo- and poly-nucleotides: 50 years of chemical synthesis. Org Biomol Chem. 2005;3(21):3851-68. PubMed PMID: 15638731.
[7] Da Ros T, Spalluto G, Prato M, Saison-Behmoaras T, Boutorine A, Cacciari B. Oligonucleotides and oligonucleotide conjugates: a new approach for cancer treatment. Curr Med Chem. 2005;12(1):71-88.
[8] Millar DP. Fluorescence studies of DNA and RNA structure and dynamics. Curr Opin Struct Biol. 1996;6(3):322-6.
[9] The Handbook. A guide to fluorescent probes and labeling technologies. Invitrogen-Molecular Probes. Ed. R. P. Haughland. Eugene, 2005. 1136 p.
[10] Dubey LV, Dubey IYa. Preparation of bifunctional silica polymer support for the synthesis of 3'-labeled oligonucleotides. Biopolym Cell. 2005; 21(4):365-9.
[11] Wachter L, Jablonski JA, Ramachandran KL. A simple and efficient procedure for the synthesis of 5'-aminoalkyl oligodeoxynucleotides. Nucleic Acids Res. 1986;14(20):7985-94.
[12] Agrawal S, Christodoulou C, Gait MJ. Efficient methods for attaching non-radioactive labels to the 5' ends of synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 1986;14(15):6227-45.
[13] Theisen P, McCullum C, Upadhya K, Jacobson K, Vu H, Andrus A. Fluorescent dye phosphoramidite labelling of oligonucleotides. Tetrahedron Lett. 1992;33(35):5033–6.
[14] Mullah B, Andrus A. Automated synthesis of double dye-labeled oligonucleotides using tetramethylrhodamine (TAMRA) solid supports. Tetrahedron Lett. 1997;38(33):5751–4.
[15] Mullah B, Livak K, Andrus A, Kenney P. Efficient synthesis of double dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assay. Nucleic Acids Res. 1998;26(4):1026-31.
[16] Korshun VA, Balakin KV, Proskurina TS, Mikhalev II, Malakhov AD, Berlin YA. A Pyrene Seco -Pseudonucleoside in Constructing Interaction-Sensitive Fluorescent DNA Probes . Nucleosides Nucleotides. 1999;18(11-12):2661–76.
[17] Stetsenko DA, Gait MJ. A convenient solid-phase method for synthesis of 3'-conjugates of oligonucleotides. Bioconjug Chem. 2001;12(4):576-86.
[18] Dubey IYa, Lyapina TV, Fedoryak DM. Microspheric silica "Silochrom-2" – a highly efficient support for solid phase oligonucleotide synthesis. Bioorg Khim. 1993; 19 (5):589-92
[19] Behrens C, Dahl O. Synthesis of achiral linker reagents for direct labelling of oligonucleotides on solid supports. Nucleosides Nucleotides. 1999;18(2):291-305.
[20] Laurent A, Debart F, Lamb N, Rayner B. Esterase-triggered fluorescence of fluorogenic oligonucleotides. Bioconjug Chem. 1997;8(6):856-61.
[21] Agrawal S, Zamecnik PC. Site specific functionalization of oligonucleotides for attaching two different reporter groups. Nucleic Acids Res. 1990;18(18):5419-23.
[22] Kahl JD, McMinn DL, Greenberg MM. High-Yielding Method for On-Column Derivatization of Protected Oligodeoxy- nucleotides and Its Application to the Convergent Synthesis of 5‘,3‘-Bis-conjugates. J Org Chem. 1998;63(15):4870–1.
[23] Dubey I, Pratviel G, Meunier B. Modification of the Thiourea Linkage of a Fluorescein?Oligonucleotide Conjugate to a Guanidinium Motif during Ammonia Deprotection. Bioconjugate Chemistry. 1998;9(5):627–32.
[24] Ju J, Ruan C, Fuller CW, Glazer AN, Mathies RA. Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis. Proc Natl Acad Sci U S A. 1995;92(10):4347-51.
[25] Glazer AN, Mathies RA. Energy-transfer fluorescent reagents for DNA analyses. Curr Opin Biotechnol. 1997;8(1):94-102.
[26] Okamura Y, Kondo S, Sase I, Suga T, Mise K, Furusawa I, Kawakami S, Watanabe Y. Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization. Nucleic Acids Res. 2000;28(24):E107.
[27] Yamane A. MagiProbe: a novel fluorescence quenching-based oligonucleotide probe carrying a fluorophore and an intercalator. Nucleic Acids Res. 2002;30(19):e97.
[28] Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. Biotechniques. 2005;39(1):75-85.
[29] Metzker ML. Emerging technologies in DNA sequencing. Genome Res. 2005;15(12):1767-76.
[30] Dirks RW, Tanke HJ. Advances in fluorescent tracking of nucleic acids in living cells. Biotechniques. 2006;40(4):489-96.
[31] Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996;14(3):303-8.
[32] Isacsson J, Cao H, Ohlsson L, Nordgren S, Svanvik N, Westman G, Kubista M, Sj?back R, Sehlstedt U. Rapid and specific detection of PCR products using light-up probes. Mol Cell Probes. 2000;14(5):321-8.
[33] Handbook of Biochemistry and Molecular Biology. Ed. G. Fasman. Boca Raton: CRC press, 1975. Vol. 1. 175 p.
[34] Sj?back R, Nygren J, Kubista M. Characterization of fluorescein-oligonucleotide conjugates and measurement of local electrostatic potential. Biopolymers. 1998;46(7):445-53.
[35] Glen Research Product Files: h
[36] Edelman LM, Cheong R, Kahn JD. Fluorescence resonance energy transfer over approximately 130 basepairs in hyperstable lac repressor-DNA loops. Biophys J. 2003;84(2 Pt 1):1131-45.