Photoaffinity Behavior of a Conjugate of Oligonucleoside Methylphosphonate, Rhodamine, and Psoralen in the Presence of Complementary Oligonucleotides

John Thaden, Paul S. Miller

Research output: Contribution to journalArticle

Abstract

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 singlestranded DNA and RNA oligonucleotides when irradiated with long-wavelength ultraviolet light. The efficiency of UV crosslinking 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.

Original languageEnglish (US)
Pages (from-to)386-394
Number of pages9
JournalBioconjugate Chemistry
Volume4
Issue number5
DOIs
StatePublished - Sep 1 1993

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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