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 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.

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

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Ficusin
Rhodamines
Oligonucleotides
Oligomers
Phosphorylation
Trioxsalen
RNA
Amines
Microscopic examination
Phosphates
DNA
Oligodeoxyribonucleotides
Single-Stranded DNA
Fluorescence
Cells
Ultraviolet Rays
Wavelength
Molecules
Microscopy
Cytoplasm

ASJC Scopus subject areas

  • Chemistry(all)
  • Organic Chemistry
  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Photoaffinity behavior of a conjugate of oligonucleoside methylphosphonate, rhodamine, and psoralen in the presence of complementary oligonucleotides. / Thaden, John; Miller, Paul S.

In: Bioconjugate Chemistry, Vol. 4, No. 5, 1993, p. 386-394.

Research output: Contribution to journalArticle

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