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Nucleic Acids Research, 2003, Vol. 31, No. 2 708-715
© 2003 Oxford University Press

Fluoride-cleavable biotinylation phosphoramidite for 5'-end-labeling and affinity purification of synthetic oligonucleotides

Shiyue Fang1,2 and Donald E. Bergstrom*,1,2

1 Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA and 2 Walther Cancer Institute, Indianapolis, IN 46208, USA

*To whom correspondence should be addressed at Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA. Tel: +1 765 494 6275; Fax: +1 765 494 9193; Email: bergstrom{at}purdue.edu

A fluoride-cleavable phosphoramidite for biotinylation was designed, synthesized and coupled efficiently to the 5'-end of DNA on an automatic synthesizer. The diisopropylsilyl acetal functionality was used to link the biotin moiety through a tertiary hydroxide group to the 5'-end of DNA. This linkage proved to be completely stable under certain post-synthetic DNA cleavage/deprotection conditions [0.05 M K2CO3 in MeOH, room temperature, 24 h and MeNH2 (~40%)/NH4OH (~29%), 1:1 v/v, 65°C, 30 min] while it can be readily broken by fluoride ion, releasing unmodified DNA. To demonstrate the use of this DNA biotinylation method, we applied this method in affinity purification of synthetic DNA. As revealed by HPLC analysis, biotinylated full-length DNA can be efficiently attached to NeutrAvidinTM coated microspheres, and failure sequences can be readily removed. Subsequent treatment of the microspheres with pyridine/HF released high quality full-length unmodified DNA in good yield.


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R. T. Pon and S. Yu
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Nucleic Acids Res., January 29, 2004; 32(2): 623 - 631.
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