Synthesis and Splice-Redirecting Activity of Branched, Arginine-Rich Peptide Dendrimer Conjugates of Peptide Nucleic Acid Oligonucleotides

Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection. Whereas 8-Arg branched peptide−PNA conjugates showed poor activity compared to a linear (R-Ahx-R)₄−PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide−PNA conjugates showed activity similar to that of the corresponding linear peptide−PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence

Confocal microscopy images of the uptake of fluorescein (FAM)-labelled CPP–PNA conjugates when incubated for 5

<p><b>Copyright information:</b></p><p>Taken from "Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent -activation in cells"</p><p>Nucleic Acids Research 2005;33(21):6837-6849.</p><p>Published online 30 Nov 2005</p><p>PMCID:PMC1301599.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>5 h with unfixed HeLa cells. Nuclei are stained red with hydroethidine. ( and ) Orange filter to view both the red colour of hydroethidine and the fluorescein fluorescence. ( and ) Green filter to view only the fluorescein fluorescence. (A and B) Show incubations in the absence of chloroquine, (C and D) Show incubations in the presence of 100 µM chloroquine. First line Tat–PNA (); second line Transportan–PNA (); third line NLS–PNA–Tat (); fourth line K–PNA (). In (C), second line, yellow dots are marked with arrows showing co-localization of hydroethidine dye and fluorescein fluorescence on the inner wall of the nucleus in several nuclei

-activation inhibitory effects of disulfide-linked CPP–PNA conjugates – in the HeLa cell reporter assay with 6 h delivery () or 24 h ()

<p><b>Copyright information:</b></p><p>Taken from "Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent -activation in cells"</p><p>Nucleic Acids Research 2005;33(21):6837-6849.</p><p>Published online 30 Nov 2005</p><p>PMCID:PMC1301599.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Firefly luciferase activity represents Tat-TAR dependent expression whilst luciferase activity represents control constitutive expression. Bars (left to right) in each case represent 2.5, 1.25, 0.625, 0.312 and 0 µM CPP–PNA concentrations

-activation inhibitory effects (firefly luciferase activity) of CPP–PNA conjugates in the HeLa cell reporter assay with delivery for 6 h in the presence of 100 µM chloroquine

<p><b>Copyright information:</b></p><p>Taken from "Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent -activation in cells"</p><p>Nucleic Acids Research 2005;33(21):6837-6849.</p><p>Published online 30 Nov 2005</p><p>PMCID:PMC1301599.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Stably-linked conjugates Tat–PNA (), TP–PNA (), TP10–PNA () and NLS–PNA–Tat (). () Disulfide-linked conjugates R–Pen-S-S–PNA (), mismatched conjugate (PNA sequence, CTCCGCAGCTCAGATC), scrambled conjugate (PNA sequence, ATCGCTCGCACCATGC), TP–S-S–PNA () and TP(int)–S-S–PNA (). Bars (left to right) in each case represent 2.5, 1.25, 0.625, 0.312 and 0 µM (light shaded bar) CPP–PNA concentrations. Control (black bar), absence of CPP–PNA and absence of chloroquine

() Secondary structure of the TAR RNA apical stem–loop, the binding site on TAR and sequence of the PNA 16mer

<p><b>Copyright information:</b></p><p>Taken from "Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent -activation in cells"</p><p>Nucleic Acids Research 2005;33(21):6837-6849.</p><p>Published online 30 Nov 2005</p><p>PMCID:PMC1301599.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Stably integrated plasmids within the HeLa Tet-Off/Tat/luc-f/luc-R cell line used for -activation inhibition studies