COMPLETE INACTIVATION OF TARGET MESSENGER-RNA BY BIOTINYLATED ANTISENSE OLIGODEOXYNUCLEOTIDE-AVIDIN CONJUGATES

Biotinylation of phosphodiester oligodeoxynucleotides (PO-ODN) allows for conjugation to avidin-based transcellular delivery systems. In add ition, biotinylation of PO-ODN at the S'-terminus provides complete pr otection against serum 3'-exonuclease degradation. The present study w as undertaken to determine if antisense 3'-biotinylated PO-ODN-avidin constructs are able to recognize and inactivate the target mRNA throug h RNase H-mediated degradation. A 21-mer antisense PO-ODN complementar y to the tat gene encompassing nucleotides 5402-5422 of the HIV-1 geno me was synthesized with biotin conjugated to the 3'-terminus (bio-tat) . Gel mobility assays using [5'-P-32]-labeled bio-tat ODN and avidin s howed that the bio-tat ODN was fully monobiotinylated. Aliquots of [P- 32]-labeled sense or antisense tat RNA (337 and 351 nucleotides, respe ctively) were prepared from transcription plasmids and were preincubat ed 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 fra gments resulting from RNase H digestion of the RNA-ODN duplex (53 and 263 nucleotides) was observed when [P-32]-tat sense RNA was incubated with antisense bio-tat ODN or conjugated to avidin or an avidin-cation ized human serum albumin (cHSA) complex. Conversely, no degradation of [P-32]-tat-antisense RNA was observed after incubation with antisense bio-tat ODN and RNase H. In addition, the avidin-cHSA complex signifi cantly increased (84-fold) the uptake of [P-32]-internally labeled bio -tat ODN and its stability against cellular nuclease degradation in pe ripheral blood lymphocytes. In conclusion, biotinylated antisense ODN- avidin constructs induce complete inactivation of target mRNA by RNase H. Therefore, 3'-biotinylated PO-ODNs have the advantages of (a) resi stance to serum and cellular 3'-exonuclease, (b) conjugation by avidin -based transcellular delivery systems, and (c) inactivation of target mRNA via RNase H degradation.