Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides.

TitleCellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides.
Publication TypeJournal Article
Year of Publication2004
AuthorsMoulton HM, Nelson MH, Hatlevig SA, Reddy MT, Iversen PL
JournalBioconjugate chemistry
Volume15
Issue2
Pagination290-9
Date Published2004 Mar-Apr
ISSN1043-1802
KeywordsAmino Acid Sequence, Animals, Arginine, Cell Membrane, Dose-Response Relationship, Drug, Hela Cells, Humans, Mice, Molecular Sequence Data, NIH 3T3 Cells, Oligonucleotides, Antisense, Peptides
Abstract

Although the sequence specificity, biostability, and low toxicity of PMO (phosphorodiamidate morpholino oligomers) make them good antisense agents to study gene function, their limited ability to cross cell membranes limits their use in cell culture. In this paper we show that conjugation to arginine-rich peptides significantly enhanced the cellular uptake of PMO. The factors that affect the conjugate's cellular uptake and its antisense activity toward a targeted mRNA were investigated. Factors studied include the number of arginines in the peptide, the choice of cross-linker, the peptide conjugation position, the length of the PMO, and the cell culture conditions. Delivery of PMO to the cell nucleus and cytosol required conjugation rather than complexation of peptides to PMO. R(9)F(2)C was best suited to deliver a PMO to its target RNA resulting in the strongest antisense effect. By simply adding the R(9)F(2)C-PMO conjugate into the cell culture medium at low microM concentration, missplicing of pre-mRNA was corrected. This particular peptide-conjugated PMO was more effective than the PMO conjugated to the transmembrane transport peptides of HIV-1 Tat protein, Drosophila antennapedia protein, or to peptides with fewer arginines. Length of PMO did not affect a peptide's delivery efficacy, but all other factors were important. R(9)F(2)C peptide provided a simple and efficient delivery of PMO to a RNA target. Conjugation of peptide to PMO enhances the opportunities to evaluate gene functions in cell cultures.

DOI10.1021/bc034221g
Alternate JournalBioconjug. Chem.