Title of article :
3-Methylphosphonate-Modified Oligo-2-O-methylribonucleotides and Their Tat Peptide Conjugates: Uptake and Stability in Mouse Fibroblasts in Culture
Author/Authors :
Prater، Chrissy E. نويسنده , , Miller، Paul S. نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2004
Abstract :
Antisense oligo-2ʹ-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 3ʹ-end of an oligo-2ʹ-O-methylribonucleotide is sufficient to prevent degradation by the 3ʹ-exonuclease activity found in mammalian serum. Complexes formed between a cationic lipid, Oligofectamine, and 5ʹ-[32P]-labeled methylphosphonate modified oligo2ʹ-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 3ʹ-methylphosphonate modified oligo-2ʹ-O-methylribonucleotides with Tat peptide, a cell permeating peptide, were also prepared. The Tat peptide was coupled to the 5ʹ-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 5ʹ-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.
Keywords :
folate , Ischaemic heart disease , homocysteine , Cretan Mediterranean diet
Journal title :
Bioconjugate Chemistry
Journal title :
Bioconjugate Chemistry