A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to a retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons.
A peptide nucleic acid (PNA) antisense for the AUG translation initiation region of prepro-oxytocin mRNA was synthesized and coupled to a r etro-inverso peptide that is rapidly taken up by cells. This bioconjugate was internalized by cultured cerebral cortex neurons within minutes, according to the specific property of the vector peptide. The PNA alone also entered the cells, but more slowly. Cell viability was unaffected when the PNA concentrations were lower than 10 microM and incubation times less than for 24 h. Magnocellular neurons from the hypothalamic supraoptic nucleus, which produce oxytocin and vasopressin, were cultured in chemically defined medium. Both PNA and vector peptide-PNA depressed the amounts of the mRNA coding for prepro-oxytocin in these neurons. A scrambled PNA had no effect and the very cognate prepro-vasopressin mRNA was not affected. The antisense PNA also depressed the immunocytochemical signal for prepro-oxytocin in this culture in a dose- and time-dependent manner. These results show that PNAs driven by the retro-inverso vector peptide are powerful antisense reagents for use on cells in culture.
CNRS-UPR 9055, Biologie des Neurones Endocrines, CCIPE, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France.
G
G
Aldrian-Herrada
M G
MG
Desarménien
H
H
Orcel
L
L
Boissin-Agasse
J
J
Méry
J
J
Brugidou
A
A
Rabié
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005822:Genetic Vectors; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D010121:Oxytocin / Q000235:genetics / Q000378:metabolism; D020135:Peptide Nucleic Acids / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D009419:Nerve Tissue Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D020135:Peptide Nucleic Acids; D011498:Protein Precursors; D012333:RNA, Messenger; D010121:Oxytocin