Complete inactivation of target mRNA by biotinylated antisense oligodeoxynucleotide-avidin conjugates.
Biotinylation of phosphodiester oligodeoxynucleotides (PO-ODN) allows for conjugation to avidin-based transcellular delivery systems. In addition, biotinylation of PO-ODN at the 3'-terminus provides complete protection against serum 3'-exonuclease degradation. The present study was undertaken to determine if antisense 3'-biotinylated PO-ODN-avidin constructs are able to recognize and inactivate the target mRNA through RNase H-mediated degradation. A 21-mer antisense PO-ODN complementary to the tat gene encompassing nucleotides 5402-5422 of the HIV-1 genome was synthesized with biotin conjugated to the 3'-terminus (bio-tat). Gel mobility assays using [5'-32P]-labeled bio-tat ODN and avidin showed that the bio-tat ODN was fully monobiotinylated. Aliquots of [32P]-labeled sense or antisense tat RNA (337 and 351 nucleotides, respectively) were prepared from transcription plasmids and were preincubated with an excess of bio-tat ODN with or without avidin constructs and digested with RNase H. Products were resolved with sequencing gel and analyzed by autoradiography. Complete conversion to predicted RNA fragments resulting from RNase H digestion of the RNA-ODN duplex (53 and 263 nucleotides) was observed when [32P]-tat sense RNA was incubated with antisense bio-tat ODN or conjugated to avidin or an avidin-cationized human serum albumin (cHSA) complex. Conversely, no degradation of [32P]-tat-antisense RNA was observed after incubation with antisense bio-tat ODN and RNase H. In addition, the avidin-cHSA complex significantly increased (84-fold) the uptake of [32P]-internally labeled bio-tat ODN and its stability against cellular nuclease degradation in peripheral blood lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Boado
R J
RJ
Department of Medicine, UCLA School of Medicine 90024.
Pardridge
W M
WM
D001360:Avidin / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects; D015756:Genes, tat / Q000187:drug effects; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects*; D016914:Ribonuclease H / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry; D014158:Transcription, Genetic
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012709:Serum Albumin; D001360:Avidin; D001710:Biotin; D016914:Ribonuclease H