Chrissy E. Prater

The HIV-1 Rev response element (RRE), a highly structured RNA sequence consisting of five stemloops, is found in all spliced and partially spliced human immunodeficiency virus (HIV) mRNA transcripts. The RRE interacts with HIV-encoded Rev protein, which facilitates exit of the transcripts from the nucleus to the cytoplasm. Because the Rev/RRE interaction is critical to virus function, it is considered a potential target for therapeutic drugs. We have investigated the interactions of antisense oligonucleotides with stem-loop II, a region that contains the high-affinity binding site for Rev. Oligo-2′-O-methylribonucleotides terminating in a nuclease resistant 3′-methylphosphonate internucleotide linkage were targeted to the 5′- or 3′-side of stem-loop IIB, which is adjacent to the Rev binding site. Thermal denaturation experiments showed that oligonucleotides of this type form highly stable duplexes with complementary single-stranded RNA. Gel electrophoretic mobility shift assays (EMSA) showed that the oligonucleotides bound with high affinity and specificity at 37°C to RRE stem-loop II RNA with apparent dissociation constants, K_D, in the low nM range. A 16-mer, 2-1mp, whose K_D is 46 nM, competitively inhibited binding of Rev peptide to RRE stem-loop II RNA as shown by EMSA experiments. When transfected into HEK 293T cells, 2-1mp inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) by 60% at a concentration of 300 nM oligonucleotide. These results are consistent with a mechanism by which 2-1mp blocks access of Rev to the RRE/CAT transcript thus preventing nuclear export and subsequent translation.

Antisense oligo-2‘-O-methylribonucleotides and their methylphosphonate derivatives show high binding affinities for their complementary targets under essentially physiological conditions. Additionally, the methylphosphonate linkage is resistant to nuclease hydrolysis. Here we show that a single methylphosphonate internucleotide linkage at the 3‘-end of an oligo-2‘-O-methylribonucleotide is sufficient to prevent degradation by the 3‘-exonuclease activity found in mammalian serum. Complexes formed between a cationic lipid, Oligofectamine, and 5‘-[³²P]-labeled methylphosphonate modified oligo-2‘-O-methylribonucleotides are taken up by mouse L₉₂₉ fibroblasts in culture. The extent of uptake appears to be dependent upon the sequence of the oligonucleotide. Examination of lysates of oligonucleotide treated cells by polyacrylamide gel electrophoresis showed that no degradation of the oligonucleotide occurred, even after incubation for 24 h. A fluorescein-derivatized oligomer was shown to localize mainly in the cell nucleus as monitored by fluorescence microscopy. Covalent conjugates of fluorescein-derivatized 3‘-methylphosphonate modified oligo-2‘-O-methylribonucleotides with Tat peptide, a cell permeating peptide, were also prepared. The Tat peptide was coupled to the 5‘-end of the oligonucleotide using either disulfide coupling chemistry or conjugation of a keto derivative of the Tat peptide via a 4-(2-aminooxyethoxy-2-(ethylureido)quinoline group at the 5‘-end of the oligonucleotide. Although formation of the Tat peptide conjugates was confirmed by mass spectrometry, the propensity of these oligonucleotides to form aggregates and their apparent high affinity for plastic and glass made the conjugates unsuitable for studies of uptake by cells in culture.