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Nucleic Acids Research, Vol 26, Issue 15 3572-3576, Copyright © 1998 by Oxford University Press

ARTICLES

Photocleavable aminotag phosphoramidites for 5'-termini DNA/RNA labeling

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

We report the design and evaluation of two non-nucleosidic photocleavable aminotag phosphor-amidites. These reagents introduce a photocleavable amino group on the 5'-terminal phosphate of synthetic oligonucleotides. The 5' photocleavable amino group enables introduction of a variety of amine-reactive markers onto synthetic oligonucleotides as well as immobilization on activated solid supports. The photocleavable bond on the 5'-phosphate can then be selectively cleaved by near-UV illumination, thereby enabling release of the marker or detachment of the oligonucleotide from a solid support. The preparation of photocleavable conjugates with biotin, digoxigenin and tetramethylrhodamine are described. In the case of biotin, a conjugate was used in a high sensitivity hybridization assay as a photocleavable probe for a complementary sequence immobilized on beads. It is also demonstrated that the 5'-PC-amino group can be used as an affinity tag for photocleavage-mediated affinity purification and phosphorylation of synthetic oligonucleotides in conjunction with activated supports. Such 5'-PC-amino labeled oligonucleotides should be useful in a variety of applications in molecular biology including multiple non-radioactive probing of DNA/RNA blots, affinity isolation and purification of nucleic acids binding proteins, diagnostic assays requiring release of the probe-target complex or specific marker, cassette mutagenesis and PCR. They will also enable the spatially-addressable photorelease of the probe-target complexes or marker molecules for diagnostic purposes.
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This article has been cited by other articles:


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X. Bai, S. Kim, Z. Li, N. J. Turro, and J. Ju
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 Proc. Natl. Acad. Sci. USAHome page
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From the Cover: Photocleavage of a 2-nitrobenzyl linker bridging a fluorophore to the 5' end of DNA
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[Abstract] [Full Text] [PDF]


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