Matrix-assisted laser desorption/ionization mass spectrometry of DNA using photocleavable biotin.
Oligonucleotides containing a photocleavable biotin (5'-PC-biotin) were analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) with wavelengths in the ultraviolet (UV) and infrared (IR) from solution and after capture on streptavidin-coated agarose or magnetic beads. The analysis was used to monitor the release of the oligonucleotides as a result of photochemical cleavage of the biotinylated linker. Near-UV pulses (UV-MALDI) led to predominant release of the photocleaved product. In contrast, only the uncleaved analyte was detected using IR pulses (IR-MALDI). Results from MALDI analysis are also presented for DNA containing a photocleavable 5'-amino group which can be covalently linked to a variety of activated surfaces and marker molecules. In a demonstration of this approach, a 5'-PC-biotinylated 49 nt RNA oligonucleotide was enzymatically synthesized using a PC-biotin-r(AG) dinucleotide primer, captured on streptavidin coated magnetic beads and analyzed by UV-MALDI. Potential applications of photocleavable linkers combined with MALDI for the analysis of nucleic acids are discussed.
Hahner
S
S
AmberGen, Inc., Boston, MA 02215, USA.
Olejnik
J
J
Lüdemann
H C
HC
Krzymañska-Olejnik
E
E
Hillenkamp
F
F
Rothschild
K J
KJ
D001483:Base Sequence; D001710:Biotin; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D007259:Infrared Rays; D010777:Photochemistry; D015202:Protein Engineering; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014466:Ultraviolet Rays
D001710:Biotin; D004247:DNA