Photoaffinity behavior of a conjugate of oligonucleoside methylphosphonate, rhodamine, and psoralen in the presence of complementary oligonucleotides.
A 3'-[[2-[N-(3-aminopropyl)-N-(2- hydroxyethyl)amino]ethylphosphoryl]oligodeoxyribonucleoside methylphosphonate 12-mer was synthesized using the Aha-CPG solid support [Thaden, J. and Miller, P.S. (1993) Bioconjugate Chem, companion paper in this issue]. The oligomer was conjugated at the 3' primary aliphatic amine with tetramethylrhodamine 5-isothiocyanate. The rhodamine linker/spacer was stable in 10% fetal calf serum. After enzymatic phosphorylation, the molecule was conjugated at the 5' phosphate with 4'-[N-(2-aminoethyl)aminomethyl]-4,5',8- trimethylpsoralen [(ae(AMT)]. The rhodamine/psoralen doubly-conjugated oligomer formed photoadducts with complementary single-stranded DNA and RNA oligonucleotides when irradiated with long-wavelength ultraviolet light. The efficiency of UV cross-linking slightly exceeded that of a colinear, psoralen-derivatized oligonucleoside methylphosphonate, and exhibited relationships with UV fluence and temperature that are characteristic for psoralen-conjugated methylphosphonates. The 1:1 complex formed with the oligodeoxyribonucleotide target could be detected by its red fluorescence. Mouse L949 cells grown in the presence of the double conjugate were shown by means of computer-assisted epifluorescence microscopy to have internalized it. There was an accumulation of intensely fluorescent points and spots in a juxtanuclear region of the cytoplasm, and a faint, diffuse signal in the entire cell area.
Thaden
J
J
Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205.
Miller
P S
PS
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000528:radiation effects; D011564:Furocoumarins / Q000737:chemistry* / Q000494:pharmacology / Q000528:radiation effects; D007091:Image Processing, Computer-Assisted; D007739:L Cells / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry* / Q000494:pharmacology / Q000528:radiation effects; D009943:Organophosphorus Compounds / Q000737:chemistry* / Q000494:pharmacology / Q000528:radiation effects; D012313:RNA / Q000737:chemistry / Q000528:radiation effects; D012235:Rhodamines / Q000737:chemistry* / Q000494:pharmacology / Q000528:radiation effects; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D011564:Furocoumarins; D009841:Oligonucleotides; D009943:Organophosphorus Compounds; D012235:Rhodamines; C032627:methylphosphonic acid; D012313:RNA