Photocleavable aminotag phosphoramidites for 5’-termini DNA/RNA labeling. Nucleic Acids Res

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 photo-cleavable 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