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Nucleic Acids Research, Vol 24, Issue 2 361-366, Copyright © 1996 by Oxford University Press

ARTICLES

Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides

J Olejnik, E Krzymanska-Olejnik and KJ Rothschild
Department of Physics and Molecular Biophysics Laboratory, Boston University, MA 02215, USA.

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.
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