Photocleavable aminotag phosphoramidites for 5'-termini DNA/RNA labeling.
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.
Olejnik
J
J
Department of Physics and Molecular Biophysics Laboratory, Boston University, Boston, MA 02215, USA.
Krzymanska-Olejnik
E
E
Rothschild
K J
KJ
D000577:Amides; D002846:Chromatography, Affinity; D004247:DNA; D005069:Evaluation Studies as Topic; D015394:Molecular Structure; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis*; D010756:Phosphoric Acids; D012313:RNA
D000577:Amides; D009841:Oligonucleotides; D010756:Phosphoric Acids; D012313:RNA; D004247:DNA; C011067:phosphoramidic acid