Loop–loop interaction of HIV-1 TAR RNA with N3′ → P5′ deoxyphosphoramidate aptamers inhibits in vitro Tat-mediated transcription

Abstract

A hairpin RNA aptamer has been identified by in vitro selection against the transactivation-responsive element (TAR) of HIV-1. A nuclease-resistant N3 3 P5 phosphoramidate isosequential analog of this aptamer also folds as a hairpin and forms with TAR a loop-loop ''kissing'' complex with a binding constant in the low nanomolar range as demonstrated by electrophoretic mobilityshift assays and surface plasmon resonance experiments. The key structural determinants, which contribute to the stability of the RNA aptamer-TAR complex, loop complementarity and the GA residues closing the aptamer loop, remain crucial for the N3 3 P5 aptamer-TAR complex. Moreover, the N3 3 P5 phosphoramidate aptamer specifically interferes with the binding of a peptide derived from the transactivator protein (Tat) peptide to TAR and selectively inhibits the Tat-mediated transcription in an in vitro assay, which marks this nuclease-resistant aptamer as a relevant candidate for experiments in cells.

Cross-Interaction between JC Virus Agnoprotein and Human Immunodeficiency Virus Type 1 (HIV-1) Tat Modulates Transcription of the HIV-1 Long Terminal Repeat in Glial Cells

“…formation of a Tat-agnoprotein complex on the interaction of Tat with TAR RNA, we performed an RNA band-shift assay using synthetic TAR RNA as a probe (22,36,41,74). 32 Plabeled TAR RNA was incubated with Tat in the absence and presence of GST and GST-agnoprotein.…”

Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex

“…Recently, chemically modified oligonucleotides directed toward the TAR stem loop, derived from the selected RNA kissing aptamer have been prepared to increase resistance to nucleases and affinity for the target (18)(19)(20). Several of these ligands have been shown to regulate TAR-dependent transcription in vitro or in cultured cells (18,21).…”

“…Several of these ligands have been shown to regulate TAR-dependent transcription in vitro or in cultured cells (18,21). Chemically modified RNA aptamers, which strongly interact with the apical loop of the TAR hairpin could then be potential candidates for the development of anti-HIV drugs able to disrupt the ternary TAR-Tat-CycT1 complex, leading to abortive RNA synthesis.…”

Regulating eukaryotic gene expression with aptamers

“…3) modifications confer resistance to nucleases and adopt the N-type (C3 0 -endo) conformation characteristic of the RNA [32]. Indeed, fully modified 2 0 -OMe and NP-DNA R06 derivatives formed complexes with TAR, characterized by similar or even slightly higher affinity constants than the parent RNA-RNA complex [33,34]. Moreover, these derivatives retained the key structural determinants identified by in vitro selection.…”

“…The biological effect of 2 0 -OMe and NP-DNA anti-TAR aptamer derivatives was evaluated. Both analogues were inhibitors of the Tat-mediated in vitro transcription with an IC 50 of about 400 nM compared to >4 lM for the in vitro selected RNA aptamer [33,34]. This effect was specific as the R06 analogue with a loop-closing C,U combination, which did not bind to TAR, did not inhibit Tat-mediated transcription.…”