Synthesis and purification of modified antisense oligodeoxyribonucleotides

Abstract

The synthesis and purification of modified antisense oligodeoxyribonucleotides (MAO) are desoribed. A new oligonucleotide purification method and a novel activating agent, dimethyl sulfoxide, for the condensation reaction are used. EDTA has been attached covalently to the 5′ end of the oligodeoxyribonucleotides sequence via an ethylenediamine linker in basic triethylammonium solution, then chelated with the metal ions such as Fe(II), Co(II) or Cu(II). High performance liquid chromatorgaphy is used to separate and purify this kind of MAO, using double mobile phase, triethylammonium acetate and acetonitrile. By using this routine chemical synthesis method, EDTA can be linked to the 5′ terminus of oligodeoxyribonucleotedes of any desired length and sequence, forming chimeric MAOs.

Keywords

oligodeoxyribonucleotedes / synthesis / purification / high performance liquid chromatography

References

[1] WeintraubH M. Antisense RNA and DNA. Sci Am, 1990, 262(1): 34-40

[2] GoodchildJ. Conjugates of oligonucleotieds and modified oligonucleotieds: a review of their synthesis and properties. Bioconjuate Chem, 1990, 1(3): 165-187

[3] SteinC A, CohenJ S. Oligodeoxynucleolides as inhibitors of gene expression: A review. Cancer Res, 1988, 48: 2659-2668

[4] Toulme’J J, HeleneC. Antimessnger oligodeoxyribonucleotides: an alternative to antisense RNA for artificial regulation of gene expression—a review. Gene, 1988, 72: 51-58

[5] GoodchildJCohenJ. Inhibition of gene expression by oligonucleotides. Oligodeoxynucleotides, Antisense Inbibition of Gene Expression, 1989, London, Macmillan Press: 53-77

[6] ChuB C F, OrgelL. Nonenzymatic sequence-specific cleavage of single-stranded DNA. Proc Natl Acad Scl USA, 1985, 82: 963-967

[7] DreyerG B, DervanP B. Sequence-specific cleavage of single-stranded DNA: Oligodeoxynucleotide-EDTA · Fe(II). Proc Natl Acad Sci USA, 1985, 82: 968-972

[8] LinS B, BlakeK R, MillerP S, et al.. Use of EDTA derivatization to characterize interactions between oligodeoxyribonucleoside methylphosphonates and nucleic acids. Biochemistry, 1989, 28: 1054-1061

[9] ThuongN T, AsselineUEcksteinF. Oligonucleotdes attached to intercalators, photoreactive and cleavage agents. Oligonucleotides and Analogues: A Practical Approach, 1991, New York, Oxford University Press: 283-306