Fluoride-cleavable biotinylation phosphoramidite for 5'-end-labeling and affinity purification of synthetic oligonucleotides.
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.
Fang
Shiyue
S
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
Bergstrom
Donald E
DE
D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005459:Fluorides / Q000737:chemistry; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D009943:Organophosphorus Compounds / Q000737:chemistry*; D019809:Streptavidin / Q000737:chemistry
D009841:Oligonucleotides; D009943:Organophosphorus Compounds; C434331:phosphoramidite; D001360:Avidin; D001710:Biotin; D019809:Streptavidin; D005459:Fluorides