3‘-Methylphosphonate-Modified Oligo-2‘-O-methylribonucleotides and Their Tat Peptide Conjugates:  Uptake and Stability in Mouse Fibroblasts in Culture

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'-[(32)P]-labeled methylphosphonate modified oligo-2'-O-methylribonucleotides are taken up by mouse L(929) 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.

Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

“…Simple peptide conjugates of oligonucleotides can be prepared in high yield and purified by RP–HPLC (30). However, cationic peptides (including many well-known CPPs) associate with negatively charged phosphodiester or phosphorothioate oligonucleotides and cause precipitation, especially when excess peptide is used to drive the reaction to completion (25,38,39). To overcome this problem, Vivès and Lebleu reported that a Tat peptide could be coupled to a deoxyoligonucleotide in the presence of 0.4 M potassium chloride solution and 40% acetonitrile to maintain solubility (38).…”

Conjugates of Oligonucleotides and Analogues with Cell Penetrating Peptides as Gene Silencing Agents

“…Another useful fragment conjugation method in solution involves oxime formation [82][83][84][85] (Fig. (9)).…”

“…A serious issue in the synthesis of oligonucleotide-based conjugates with highly cationic (positively charged) peptides, which includes many CPPs, is that simple mixing of an oligonucleotide and a cationic peptide may lead to irreversible precipitation of the resultant conjugate and/or complex [65,85]. To overcome the problem, organic co-solvents [65] or non-ionic denaturing agents need to be added [63,66,69].…”

Advanced Microfluorescence Methods in Monitoring Intracellular Uptake of “Antisense” Oligonucleotides

“…Also mouse cell lines are quite often used, e.g. B16 -melanoma cell line [40], RAW264.7 -monocyte macrophage cell line [79], L929 -fibroblast cells [83], or MCA207 -chemically induced murine fibrosarcoma [40].…”