Antisense oligonucleotides directed to the HIV-1 Rev Response Element: Effects of nucleotide modification on oligo binding affinity, nuclease stability, and cellular uptake

This thesis describes the design, synthesis, and characterization of antisense oligonucleotides aimed at the targeted disruption of the high-affinity Rev binding site on stem IIB of the HIV-1 Rev Response Element (RRE) RNA. Several nucleotide modifications were studied for their potential to increase binding affinity with the RRE RNA target and impart nuclease resistance to anti-RRE oligos. Two steric-hindrance oligonucleotide sequences containing 2'-O-methyl sugars and single methylphosphonate internucleotide linkages were found to be nuclease resistant in an in vitro serum digest assay and during 24-hour incubation in mouse fibroblast cells in culture. These sequences were shown to have apparent dissociation constants in the low nM range, indicating high binding-affinity of these modified oligos for their RRE RNA target. We found that oligo 2-1mp, a steric-hindrance oligonucleotide, can prevent Rev peptide binding when the oligo is pre-bound to the RRE target---presumably by disrupting the structure of stem IIB and the high-affinity Rev binding site. Moreover, when added simultaneously with Rev-peptide, oligo 2-1mp can compete for RRE binding. Oligo 2-1mp is also capable of competition when Rev-peptide is pre-bound to the RRE.; In addition to structural disruption of RRE IIB with oligo 2-1mp , we have also investigated the potential for cellular RNAse H activation and RRE target destruction by designing chimeric oligonucleotides containing six or eight 2'-deoxy residues in the center of this sequence. The duplex-stabilizing modifications 5-propynyl C and U were also investigated for their ability to increase chimera/RNA binding affinity and activate RNAse H-mediated RNA hydrolysis. All deoxy- and deoxy-propynyl chimeras were found to be RNAse H competent, and positions of RNAse H cleavage of the RRE target have been mapped. We found however, that the incorporation of 5-propynyl C and U residues in the deoxy chimeras does not substantially increase binding affinity for RRE IIB, and can be detrimental to the RNAse H competency of the oligonucleotides. Oligos 2-Imp and the 6-deoxy chimera are currently being tested for anti-HIV activity and the ability to inhibit Rev-mediated nuclear export in cell culture.