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The synthesis and characterization of photocleavable peptide-DNA conjugates is described along with their use as photocleavable mass marker (PCMM) hybridization probes for the detection of target DNA sequences by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Three photocleavable peptide-DNA conjugates were synthesized, purified, and characterized using HPLC and denaturing gel electrophoresis, as well as IR-MALDI and UV-MALDI. The hybridization properties of the conjugates were also studied by monitoring their thermal denaturation with absorption spectroscopy. No significant difference in the melting temperature ( T (m)) of the duplexes was observed between the unmodified duplex and the duplex in which one strand was modified with the photocleavable peptide moiety. These conjugates were evaluated as hybridization probes for the detection of immobilized synthetic target DNAs using MALDI-MS. In these experiments, the DNA portion of the conjugate acts as a hybridization probe, whereas the peptide is photoreleased during the ionization/desorption step of UV-MALDI and can serve as a marker (mass tag) to identify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.

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.

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