Biopolym. Cell. 2007; 23(2):137-142.
Біоорганічна хімія
Синтез 3'- та 3,5-модифікованих олігонуклеотидів на функціоналізованому силікагелі «Силохром-2»
1Дубей І. Я., 1Дубей Л. В., 2Федоряк Д. М.
  1. Інститут молекулярної біології і генетики НАН України
    Вул. Академіка Заболотного, 150, Київ, Україна, 03680
  2. Інститут біоорганічної хімії та нафтохімії НАН України
    вул. Мурманська, 1, Київ, Україна, 02094


Описано отримання З'-моно- та 3' ,5' -біс-мічених олігонуклеотидів на новому полімерному носієві з використанням мікросферичного силікагелю «Силохром-2», функціоналізованому флуоресцеїном. На цьому полімері твердофазним фосфітамідним методом синтезовано олігонуклеотид (dT)15, шр містить залишок барвника на 3'-кінці Постсинтетичною 5'-модифікацією З'-конюгата вдалося отримати олігомер, мічений двома молекулами флуоресцеїну. У майбутньому це буде використано для синтезу флуоресцентних зондів для детекції нуклеїнових кислот.
Keywords: олігонуклеотидні кон'югати, флуоресцентні мітки, твердофазний синтез, функціоналізація


[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.