Journal Article

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

Jerzy Olejnik, Edyta Krzymanska-Olejnik and Kenneth J. Rothschild

in Nucleic Acids Research

Volume 26, issue 15, pages 3572-3576
Published in print August 1998 | ISSN: 0305-1048
Published online August 1998 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/26.15.3572
Photocleavable aminotag phosphoramidites for 5′-termini DNA/RNA labeling

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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.

Journal Article.  2782 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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