3′-methylphosphonate-modified oligo-2′-O-methylribonucleotides and their Tat peptide conjugates: Uptake and stability in mouse fibroblasts in culture

Chrissy E. Prater, Paul S. Miller

Research output: Contribution to journalArticle

Abstract

Antisense oligo-2′-O-methylribonucleotides and their methylphosphonate derivatives show high binding affinities for their complementary targets under essentially physiological conditions. Additionally, the methylphosphonate linkage is resistant to nuclease hydrolysis. Here we show that a single methylphosphonate internucleotide linkage at the 3′-end of an oligo-2′-O-methylribonucleotide is sufficient to prevent degradation by the 3′-exonuclease activity found in mammalian serum. Complexes formed between a cationic lipid, Oligofectamine, and 5′-[32P]-labeled methylphosphonate modified oligo2′-O-methylribonucleotides are taken up by mouse L929 fibroblasts in culture. The extent of uptake appears to be dependent upon the sequence of the oligonucleotide. Examination of lysates of oligonucleotide treated cells by polyacrylamide gel electrophoresis showed that no degradation of the oligonucleotide occurred, even after incubation for 24 h. A fluorescein-derivatized oligomer was shown to localize mainly in the cell nucleus as monitored by fluorescence microscopy. Covalent conjugates of fluorescein-derivatized 3′-methylphosphonate modified oligo-2′-O-methylribonucleotides with Tat peptide, a cell permeating peptide, were also prepared. The Tat peptide was coupled to the 5′-end of the oligonucleotide using either disulfide coupling chemistry or conjugation of a keto derivative of the Tat peptide via a 4-(2-aminooxyethoxy-2-(ethylureido)quinoline group at the 5′-end of the oligonucleotide. Although formation of the Tat peptide conjugates was confirmed by mass spectrometry, the propensity of these oligonucleotides to form aggregates and their apparent high affinity for plastic and glass made the conjugates unsuitable for studies of uptake by cells in culture.

Original languageEnglish (US)
Pages (from-to)498-507
Number of pages10
JournalBioconjugate Chemistry
Volume15
Issue number3
DOIs
StatePublished - May 2004

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Oligonucleotides
Fibroblasts
Cell culture
Peptides
spleen exonuclease
Fluorescein
Cells
Derivatives
Degradation
Fluorescence microscopy
Polyacrylates
Electrophoresis
Cell Nucleus
Fluorescence Microscopy
Oligomers
Disulfides
Lipids
Plastics
Glass
Mass spectrometry

ASJC Scopus subject areas

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

Cite this

3′-methylphosphonate-modified oligo-2′-O-methylribonucleotides and their Tat peptide conjugates : Uptake and stability in mouse fibroblasts in culture. / Prater, Chrissy E.; Miller, Paul S.

In: Bioconjugate Chemistry, Vol. 15, No. 3, 05.2004, p. 498-507.

Research output: Contribution to journalArticle

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