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

Abstract

Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R9F2 was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R9 conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R9F2 and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide.

The Biological Effect of an Antisense Oligonucleotide Depends on Its Route of Endocytosis and Trafficking

“…In both cases the fl uorescent oligonucleotide was primarily found in intracellular vesicles, similar to previous descriptions of the subcellular distribution of oligonucleotides and peptide-oligonucleotide conjugates observed by us and by others (Turner et al, 2005;Alam et al, 2008). Thus, we sought to probe possible differences in uptake and traffi cking pathways by use of several putatively selective inhibitors of various endocytotic pathways (Parpal et al, 2001;Khalil et al, 2006).…”

“…It should be noted that the uptake studies described here track the accumulation and retention of the fl uorophore; thus, it is possible that this measurement might not refl ect accumulation of the oligonucleotide itself if the labels were to be cleaved. However, it has been our experience Kang et al, 2008) and that of several other laboratories (Turner et al, 2005;Bendifallah et al, 2006;El-Andaloussi et al, 2006) that fl uorophore labels placed at the 3′ position of stable oligonucleotides provide reasonably accurate tracking of oligonucleotides in cell culture experiments such as those described here.…”

Cell‐penetrating peptides: Nanocarrier for macromolecule delivery in living cells

“…A simple one-step method for cotransduction of sleeping beauty transposase and donor plasmid using Tat has been demonstrated (69). Oligonucleotide analogs (LNAs) conjugated to CPPs, such as Tat, penetratin, TP, and Arg9F2, were internalized in the cells but failed to show any biological activity because of endosomal entrapment (75). However, PNA-CPP conjugates were released from the endosomal trap by addition of chloroquine (76).…”

Marked transfection enhancement by the DPL (DNA/peptide/lipid) complex

“…The Tat peptide was initially synthesized with a C-terminal cysteine modification for chemical conjugation to cargo DNA molecules for improved transfection. Transfection enhancement has been reported with chemical conjugates of a peptide to oligonucleotides (31,32). In one series of the experiments, the Tat peptide with the C-terminal modification was simply combined with DNA and then with cationic lipids, and compared for transfection efficiency as a control.…”