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. 2003 Jan 15;31(2):708-15.
doi: 10.1093/nar/gkg130.

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

Shiyue Fang  1 , Donald E Bergstrom
Affiliations

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

Shiyue Fang et al. Nucleic Acids Res. .

Abstract

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 K(2)CO(3) in MeOH, room temperature, 24 h and MeNH(2) (approximately 40%)/NH(4)OH (approximately 29%), 1:1 v/v, 65 degrees 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 NeutrAvidin 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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Figures

Figure 1
Figure 1
HPLC traces. (a) Crude DNA obtained by 0.05 M K2CO3/MeOH cleavage/deprotection. (b) After treating with pyridine/HF. (c) After affinity purification. (d) Unbiotinylated failure sequences left after NeutrAvidin™ treatment. (e) Control DNA and affinity purified DNA. (f) Affinity purified DNA using PBS as buffer. (g) Control DNA. (h) DMTr-selective cartridge purified DNA. (i) Crude DNA obtained by MeNH2 (40%)/NH4OH (29%) (1:1 v/v) cleavage/deprotection. (j) After treating with pyridine/HF.
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Scheme 2.
See this image and copyright information in PMC

References

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