title: Complete protection of antisense oligonucleotides against serum nuclease degradation by an avidin-biotin system.
abstract: It has been recently demonstrated that a complex of avidin, a cationic protein, and a monobiotinylated antisense oligonucleotide for the GLUT1 glucose transporter mRNA is taken up by cells in vitro and by organs in vivo via absorptive-mediated endocytosis. In the present study, a GLUT1 biotinylated oligonucleotide-avidin construct showing complete protection against serum 3'-exonuclease-mediated degradation is described. 21-mer antisense oligonucleotides complementary to nucleotides 162-182 and 161-181 of the bovine GLUT1 glucose transporter mRNA were synthesized with a 6-aminodeoxyuridine at positions 3 and 20, respectively, biotinylated with NHS- or NHS-XX-biotin to yield near 5'- or near 3'-biotinylated oligonucleotide (bio-DNA), and 5'- and 3'-end radiolabeled. Serum induced a rapid degradation of unprotected (no avidin) [5'-32P]-5'-bio-DNA (> 95% at 30 min). Avidin partially protected this construct (approximately 31% of intact 21-mer oligo remained at 1 h). Similar results were obtained with the [3'-32P]-5'-bio-DNA; however, no degradation products of varying size were observed, confirming that the degradation is mediated primarily by a 3'-exonuclease. Incubation of the [5'-32P]-3'-bio-DNA with serum showed a rapid conversion to the 20- and 19-mer forms (t1/2 approximately 13 min). Conversely, avidin totally protected this construct against the serum 3'-exonuclease. In conclusion, avidin fully protects antisense oligonucleotides biotinylated at the near 3'-terminus against serum 3'-exonuclease degradation, and this property may be useful for avidin-mediated drug delivery of oligonucleotides to tissues in vivo or to cultured cells in vitro.
authors.affiliation: Department of Medicine, UCLA School of Medicine 90024.
authors.forename: R J
authors.initials: RJ
authors.lastname: Boado
authors.forename: W M
authors.initials: WM
authors.lastname: Pardridge
mesh_terms: D000818:Animals; D001360:Avidin / Q000031:analogs & derivatives / Q000494:pharmacology*; D001483:Base Sequence; D001710:Biotin / Q000031:analogs & derivatives / Q000494:pharmacology*; D005092:Exonucleases / Q000037:antagonists & inhibitors / Q000097:blood*; D051272:Glucose Transporter Type 1; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats
chemical_list: D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C495679:Slc2a1 protein, rat; D001360:Avidin; D001710:Biotin; D005092:Exonucleases