Document 0727
 DOCN  M9550727
 TI    Complete inactivation of target mRNA by biotinylated antisense
       oligodeoxynucleotide-avidin conjugates.
 DT    9505
 AU    Boado RJ; Pardridge WM; Department of Medicine, UCLA School of Medicine
       90024.
 SO    Bioconjug Chem. 1994 Sep-Oct;5(5):406-10. Unique Identifier : AIDSLINE
       MED/95151809
 AB    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)
 DE    Avidin/CHEMISTRY/*PHARMACOLOGY  Base Sequence
       Biotin/CHEMISTRY/*PHARMACOLOGY  Endocytosis/DRUG EFFECTS  Genes,
       tat/DRUG EFFECTS  Genome, Viral  Human  HIV-1/GENETICS  In Vitro
       Lymphocytes/DRUG EFFECTS/METABOLISM  Molecular Sequence Data
       Oligonucleotides, Antisense/CHEMISTRY/*PHARMACOLOGY  Ribonuclease H,
       Calf Thymus/METABOLISM  RNA, Messenger/*DRUG EFFECTS  Serum
       Albumin/CHEMISTRY  Support, U.S. Gov't, P.H.S.  Transcription, Genetic
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).