Bent Pseudoknots and Novel RNA Inhibitors of Type 1 Human Immunodeficiency Virus (HIV-1) Reverse Transcriptase

Inhibition of HIV-1 reverse transcriptase by RNA aptamers in Escherichia coli

“…This design was intended to minimize nucleolytic degradation and to maximize the probability that nascent transcripts would present the aptamers to the intracellular environment in their native conformations. Nitrocellulose filter-binding assays of transcripts synthesized in vitro with these flanking sequences gave Kd values (Kd TPK1.1 = 1.1 nM; Kd 70.28 = 5.3 nM; Kd 70.28min = 25 nM; Kd 70.24 = 43 nM) that are similar to those originally reported for the core aptamers (Tuerk et al 1992;Burke et al 1996). Aptamer expression plasmids were introduced into BK148 harboring plasmid pRT5.…”

“…As noted above, RNA aptamers show potent antiviral activity in human cell culture. We have speculated that HIV-1 RT may not be able to mutate to aptamer resistant forms without adversely affecting viral replication activity (Burke et al 1996). This hypothesis appears to have been born out in at least one instance (Fisher et al 2002).…”

“…1). Aptamers 70.28 and 70.24 are 118-nt isolates selected to bind HIV-1 RT, whereas 70.28min is the 34-nt pseudoknot core of isolate 70.28 (Burke et al 1996). Aptamer TPK1.1 is a related 33-nt pseudoknot aptamer (Tuerk et al 1992), which has been reported to bind HIV-1 RT in solution with enhanced affinity (Kd ≈ 25 pM; Kensch et al 2000).…”

“…Cells carrying plasmid pRT(p), an RT-overexpressing plasmid (gift of S. Hughes, National Cancer Institute), served as positive control for RT activity. Dissociation constants (Kd) were determined from nitrocellulose filter-binding assays as described (Burke et al 1996) using gel-purified, radiolabeled aptamer RNA and purified HIV-1 RT carrying a His 6 tag on its carboxyl terminus.…”

“…However, drug-resistant forms of RT are selected by prolonged treatment, prompting widespread efforts to generate new anti-RT drugs. Both RNA and ssDNA aptamers that bind HIV-1 RT with high affinity have been identified through the SELEX protocol (Tuerk et al 1992;Schneider et al 1995;Burke et al 1996;Andreola et al 2001). When any of several different RNA aptamers was expressed in human T293 cells, the susceptibility of these cells to HIV infection was reduced, and the infectivity of viruses produced by infected T293 cells is reduced 90%-99% compared with cells not expressing aptamers .…”