Development of sensitive radioiodinated anti-sense oligonucleotide probes by conjugation technique

Mrinal K. Dewanjee, Abdol K. Ghafouripour, Rudolf K. Werner, Aldo N Serafini, George N Sfakianakis

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Radioactive and nonradioactive oligonucleotide (ON) probes have been used in a variety of studies of in vitro hybridization for locations of specific genes and determination of the level of mRNA transcription in activated and proliferative cells. Nuclease-resistant phosphorothioate derivatives of oligonucleotides are also finding potential therapeutic applications in intact cells and animal models. In this study, we have developed new techniques of radioiodination by the conjugation of oligonucleotides with p-methoxyphenyl isothiocyanate (PMPITC) and optimized the conditions of the radioiodination, e.g. concentration of reactants, oxidants, pH, time, and temperature. We have used a 25-mer actin mRNA probe as a model substrate for radioiodination. The ON probe was modified by conjugation with the addition of aminohexyl (AH) group at the 5′-terminal. Both the techniques of preiodination of PMPITC followed by conjugation and radioiodination of PMPITC-conjugated AHON were evaluated; the latter was found to be more convenient. The optimum pH of radioiodination of PMPITC with 125I was 6.5; on the other hand, the optimum pH for PMPITC-conjugated AHON was 8.5. The conditions of conjugation were optimized with 125I-labeled PMPITC. The techniques of paper chromatography and HPLC with a Spherogel column were developed for the purification and characterization of these oligonucleotide probes. Without PMPITC conjugation, the yield of direct radioiodination of the same oligonucleotide was very low (4-6%); the yield in our optimized technique is 50-60%. Radioiodination of conjugated oligonucleotides is a simple and efficient route to radiolabeled probes for hybridization studies.

Original languageEnglish
Pages (from-to)195-200
Number of pages6
JournalBioconjugate Chemistry
Volume2
Issue number4
StatePublished - Dec 1 1991

Fingerprint

Oligonucleotide Probes
Antisense Oligonucleotides
Oligonucleotides
Phosphorothioate Oligonucleotides
Paper Chromatography
Messenger RNA
Distillation columns
Transcription
Chromatography
isothiocyanic acid
Oxidants
Purification
Actins
Animals
Animal Models
Genes
High Pressure Liquid Chromatography
Derivatives
Temperature
Substrates

ASJC Scopus subject areas

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

Cite this

Dewanjee, M. K., Ghafouripour, A. K., Werner, R. K., Serafini, A. N., & Sfakianakis, G. N. (1991). Development of sensitive radioiodinated anti-sense oligonucleotide probes by conjugation technique. Bioconjugate Chemistry, 2(4), 195-200.

Development of sensitive radioiodinated anti-sense oligonucleotide probes by conjugation technique. / Dewanjee, Mrinal K.; Ghafouripour, Abdol K.; Werner, Rudolf K.; Serafini, Aldo N; Sfakianakis, George N.

In: Bioconjugate Chemistry, Vol. 2, No. 4, 01.12.1991, p. 195-200.

Research output: Contribution to journalArticle

Dewanjee, MK, Ghafouripour, AK, Werner, RK, Serafini, AN & Sfakianakis, GN 1991, 'Development of sensitive radioiodinated anti-sense oligonucleotide probes by conjugation technique', Bioconjugate Chemistry, vol. 2, no. 4, pp. 195-200.
Dewanjee, Mrinal K. ; Ghafouripour, Abdol K. ; Werner, Rudolf K. ; Serafini, Aldo N ; Sfakianakis, George N. / Development of sensitive radioiodinated anti-sense oligonucleotide probes by conjugation technique. In: Bioconjugate Chemistry. 1991 ; Vol. 2, No. 4. pp. 195-200.
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