Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS.
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.
Olejnik
J
J
AmberGen Inc., 1106 Commonwealth Avenue, Boston, MA 02215, USA. jerzy@ambergen.com
Lüdemann
H C
HC
Krzymanska-Olejnik
E
E
Berkenkamp
S
S
Hillenkamp
F
F
Rothschild
K J
KJ
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes; D006868:Hydrolysis; D009693:Nucleic Acid Hybridization; D010455:Peptides / Q000737:chemistry*; D010777:Photochemistry; D011489:Protein Denaturation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D015342:DNA Probes; D010455:Peptides; D004247:DNA