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Targeting of antisense PNA oligomers to human galanin receptor type 1 mRNA
Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. Tartu University, Estonia.
Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
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2004 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 38, no 5, 316-324 p.Article in journal (Refereed) Published
Abstract [en]

In this work, we have targeted positions 18–38 of the human galanin receptor type 1 (GalR1) mRNA coding sequence with different peptide nucleic acid (PNA) oligomers. This region has previously been shown to be a good antisense region and therefore we aimed to identify the subregions and/or thermodynamic parameters determining the antisense efficacy. Nine different PNA oligomers were conjugated to a cell-penetrating peptide, transportan, to enhance their cellular uptake. Concentration-dependent down-regulation of GalR1 protein expression in human melanoma cell line Bowes was measured by radioligand binding assay. No reduction of GalR1 mRNA level was observed upon PNA treatment, thus, the effect was concluded to be translational arrest. Judging from the EC50 values, antisense PNA oligomers targeting regions 24–38 (EC50 = 70 nM) or 27–38 (EC50 = 80 nM) were the most potent suppressors of protein expression. No parameter predicted by M-fold algorithm was found to correlate with the measured antisense activities. Presence of some subregions was found not to increase antisense efficiency of PNA. Presence of a short unpaired triplet between nucleotides 33 and 35 in the target region was, on the other hand, found to be the most critical for efficient GalR1 down-regulation. Thus, the results are of high impact in designing antisense oligomers. Specific results of this study demonstrate 20-fold more efficient antisense down-regulation of GalR1 as achieved before.

Place, publisher, year, edition, pages
2004. Vol. 38, no 5, 316-324 p.
Keyword [en]
antisense, cell penetrating peptide, peptide nucleic acid, M-fold algorithm
National Category
URN: urn:nbn:se:su:diva-23481DOI: 10.1016/j.npep.2004.06.005ISI: 000224748100007OAI: diva2:192276
Available from: 2004-11-25 Created: 2004-11-25 Last updated: 2015-04-22Bibliographically approved
In thesis
1. Cell-penetrating peptides and bioactive cargoes: Strategies and mechanisms
Open this publication in new window or tab >>Cell-penetrating peptides and bioactive cargoes: Strategies and mechanisms
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell membrane is an impermeable barrier for most macromolecules. Recently discovered cell-penetrating peptides (CPPs) have gained lot of attention because they can cross the membrane, and even more, carry cargoes with them. How CPPs enter cells is still not clear, while the delivery of different cargoes has been convincingly shown. This thesis concentrates on evaluating CPPs as vectors for different biologically relevant cargoes. Proposed internalisation mechanisms are reviewed as well as cargo coupling strategies. Biological activities of antisense oligonucleotides delivered by CPPs have been of particular interest and are explained in greater details.

A new CPP, pIsl, was derived from Islet-1 transcription factor, and compared to archetypical CPPs like penetratin and transportan. All three peptides resided in the headgroup region of lipid bilayers in model membranes. However, penetratin and pIsl did only interact with negatively charged membranes, while transportan did not distinguish negatively charged and neutral membranes. This suggests different translocation pathways for different CPPs. Biotinylated pIsl and penetratin were complexed with avidin, and uptake of avidin into the human melanoma cell line Bowes was observed in both cases. This means that the protein is not unfolded during the translocation process, which is important in delivery of other, biologically active proteins.

Transportan and its analogue TP10 were used for peptide nucleic acid (PNA) antisense oligonucleotide delivery. First, eight human galanin receptor type 1 targeting PNA oligomers were designed, conjugated to transportan and assayed for antisense efficiency. In contrary to avidin-biotinylated peptide conjugate, a covalent bond between PNA oligomers and the transport peptide was necessary for cellular uptake of oligomers. A common problem in antisense technology is inactivity of antisense oligonucleotides due to the secondary structure of the target. Efficiencies of tested galanin receptor type 1 targeting PNA oligomers varied over two orders of magnitude. The most efficient oligomers were targeting coding sequence regions 24-38 and 27-38, and had EC50 values 70 and 80 nM, respectively.

TP10-antisense PNA oligomer conjugates were targeted also to L-type voltage dependent Ca2+ channel subunits CaV1.2 and CaV1.3. Specific down-regulation of respective proteins was demonstrated by immunohistochemistry. Physiological response to the down-regulation of either of Ca2+ channels was studied by alteration of flexor reflex sensitisation. Rats treated with either of the antisense PNA, but not with scrambled PNA lost the action potential windup phenomenon. In conjunction with a variety of drugs, modulating the conductivity and excitability of neuronal membranes, a central role of L-type CaV channels in sensitisation was confirmed. Nevertheless, also N-methyl-D-aspartate and glycine receptors were found to be required.

Finally, delivery of plasmids by TP10 was evaluated. In contrary to many similar CPPs, TP10 was incapable to translocate plasmids to cells. However, addition of TP10 or a TP10-PNA conjugate to polyethyleneimine-condensed plasmids increased the expression of reporter genes.

In summary, different types of cargoes have been delivered by CPPs and different cargo coupling strategies have been used. CPP-mediated antisense oligonucleotide delivery has been used to identify accessible sites in human galanin receptor type 1 mRNA and to determine the role of L-type voltage dependent Ca2+ channels in axon potential windup.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2004. 75 p.
Cell-penetrating peptide, delivery, Peptide nucleic acid
National Category
Biochemistry and Molecular Biology
urn:nbn:se:su:diva-308 (URN)91-7265-986-6 (ISBN)
Public defence
2004-12-17, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Available from: 2004-11-25 Created: 2004-11-25 Last updated: 2015-03-20Bibliographically approved

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Langel, Ülo
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