Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides.
We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5'-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300-350 nm light the 5'-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5'-phosphorylated oligonucleotides into solution. 5'-PCB-labeled oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB-phosphoramidite has been used for the synthesis, purification and phosphorylation of 50-and 60mer oligonucleotides.
Olejnik
J
J
Department of Physics and Molecular Biophysics Laboratory, Boston University, MA 02215, USA.
Krzymańska-Olejnik
E
E
Rothschild
K J
KJ
D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D010766:Phosphorylation; D010782:Photolysis; D014466:Ultraviolet Rays
D009841:Oligonucleotides; D009943:Organophosphorus Compounds; C098492:biotin phosphoramidite; D001710:Biotin