TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein.
One approach to investigate gene function, by silencing the activity of certain proteins, is the usage of double stranded decoy oligodeoxynucleotides (ds decoy ODNs). Decoy, in this sense, is ds ODNs bearing the consensus binding sequence for a DNA-binding protein. This can be used in clinical settings to attenuate the effect of overexpressed transcription factors in tumor cells. We here choose to target the oncogenic protein Myc. Since oligonucleotides are poorly internalized to cells, a cell-penetrating peptide, TP10, was coupled to the Myc decoy, using two different strategies. Either TP10 was simply mixed with ds decoy ODNs forming complexes through non-covalent electrostatic interactions, or by having a nona-nucleotide overhang in one of the decoy strands, and adding a complementary PNA sequence coupled to an NLS sequence and TP10, which could hybridize to the Myc decoy. By using these strategies, uptake was significantly enhanced, especially with the co-incubation approach. Interestingly, various endocytosis inhibitors had no effect on the uptake pattern, suggesting that uptake of these complexes is not mediated via endocytosis. Finally, a decreased proliferative capacity was observed when treating the neuroblastoma cell line N2a with TP10-PNA conjugate hybridized to Myc decoy compared to naked Myc decoy and untreated cells. A dose-dependent decrease in proliferation was also observed in MCF-7 cells, when using both strategies. These results suggest an alternative way to efficiently deliver ds ODNs into cells using the cell-penetrating peptide TP10 and prevent tumor growth by targeting the oncogenic protein Myc.
El-Andaloussi
S
S
Department of Neurotoxicology, Stockholm University, Sweden. samir@neurochem.su.se
Johansson
H
H
Magnusdottir
A
A
Järver
P
P
Lundberg
P
P
Langel
Ulo
U
D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016503:Drug Delivery Systems; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000008:administration & dosage* / Q000378:metabolism; D020135:Peptide Nucleic Acids / Q000008:administration & dosage* / Q000235:genetics; D010455:Peptides / Q000008:administration & dosage* / Q000235:genetics; D010957:Plasmids; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000378:metabolism
D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; C516255:TP10-PNA conjugate