Conjugates of antisense oligonucleotides with the Tat and antennapedia cell-penetrating peptides: effects on cellular uptake, binding to target sequences, and biologic actions.
OBJECTIVE
The attainment of effective intracellular delivery remains an important issue for pharmacologic applications of antisense oligonucleotides. Here, we describe the synthesis, binding properties, and biologic properties of peptide-oligonucleotide conjugates comprised of the Tat and Ant cell-penetrating peptides with 2'-O-methyl phosphorothioate oligonucleotides.


METHODS
The biologic assay used in this study measures the ability of the antisense molecule to correct splicing of an aberrant intron inserted into the Luciferase gene; thus, this assay clearly demonstrates the delivery of functional antisense molecules to the splicing machinery within the nucleus. The binding affinities of the conjugates to their target sequences were measured by surface plasmon resonance (BIAcor) techniques.


RESULTS
The peptide-oligonucleotide conjugates progressively entered cells over a period of hours and were detected in cytoplasmic vesicles and in the nucleus. Peptide-oligonucleotide conjugates targeted to the aberrant splice site, but not mismatched controls, caused an increase in Luciferase activity in a dose-responsive manner. The kinetics of Luciferase appearance were consistent with the course of the uptake process for the conjugates. The effects of peptide conjugation on the hybridization characteristics of the oligonucleotides were also examined using surface plasmon resonance. The peptide-oligonucleotide conjugates displayed binding affinities and selectivities similar to those of unconjugated oligonucleotides.


CONCLUSIONS
Conjugation with cell-penetrating peptides enhances oligonucleotide delivery to the nucleus without interfering with the base-pairing function of antisense oligonucleotides.
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599, USA.
Anna
A
Astriab-Fisher
Dimitri
D
Sergueev
Michael
M
Fisher
Barbara Ramsay
BR
Shaw
R L
RL
Juliano
D000595:Amino Acid Sequence; D051943:Antennapedia Homeodomain Protein; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D016503:Drug Delivery Systems / Q000379:methods; D015696:Gene Products, tat / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics*; D006131:Growth Inhibitors / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D018398:Homeodomain Proteins / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics*; D011485:Protein Binding / Q000235:genetics; D014157:Transcription Factors
D051943:Antennapedia Homeodomain Protein; D015696:Gene Products, tat; D006131:Growth Inhibitors; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D014157:Transcription Factors