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

• Published in: Nucleic acids research Vol. 33; no. 1; pp. 27 - 42
• Main Authors: Turner, John J., Arzumanov, Andrey A., Gait, Michael J.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-01-2005; Oxford Publishing Limited (England)
• Subjects: Biological Transport; Cell Membrane - metabolism; Cells, Cultured; Disulfides - chemistry; Fibroblasts - metabolism; Gene Products, tat - antagonists & inhibitors; HeLa Cells; HIV Long Terminal Repeat - drug effects; HIV-1 - genetics; Humans; Hydrophobic and Hydrophilic Interactions; Oligonucleotides - chemical synthesis; Oligonucleotides - metabolism; Oligonucleotides - pharmacology; Oligonucleotides, Antisense - chemical synthesis; Oligonucleotides, Antisense - chemistry; Oligonucleotides, Antisense - pharmacology; Peptide Nucleic Acids - chemical synthesis; Peptide Nucleic Acids - metabolism; Peptide Nucleic Acids - pharmacology; tat Gene Products, Human Immunodeficiency Virus; Thionucleotides - chemical synthesis; Thionucleotides - chemistry; Thionucleotides - pharmacology; Transcriptional Activation - drug effects
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gki142

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.