A Structure-Activity Study of the Inhibition of HIV-1 Tat-DependentTrans-Activation by Mixmer 2′-O-Methyl Oligoribonucleotides Containing Locked Nucleic Acid (LNA),α-L-LNA, or 2′-Thio-LNA Residues

Abstract

The HIV-1 trans-activation responsive element (TAR) RNA stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibitors of these interactions block full-length transcription and, hence, would potentially inhibit HIV replication. We have studied structure-activity relationships in inhibition of trans-activation by steric block 2'-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. Inhibition was measured both in Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract and in a robust HeLa cell reporter assay that involves use of stably integrated plasmids to express firefly luciferase Tat dependently and Renilla luciferase Tat-independently. OMe oligonucleotides with optimally 40%-50% LNA units and a minimum of 12 residues in length were active in the cellular assay when delivered with cationic gemini surfactant GS11 at 50% inhibitory concentrations of 230 +/- 40 nM, whereas activity in the in vitro transcription assay was observed down to 9 residues. No cellular activity was observed for OMe oligonucleotides of 12 or 16 residues, which was shown to be due to poor cellular uptake. Both 12-mer mixmers containing alpha -L-LNA or 2'-thio-LNA (S-LNA) were also active in in vitro transcription and the former in cellular reporter inhibition assays, demonstrating that the property of promotion of cellular uptake by LNA is not due to specific sugar conformational effects. Covalent conjugates of OMe/LNA chimeras with Kaposi-fibroblast growth factor (K-FGF) or Transportan peptides failed to enter HeLa cells without a delivery agent but were fully active when delivered by cationic gemini surfactant, showing that in principle, peptide conjugation does not interfere with cellular activity. Thus, OMe/LNA mixmers are powerful reagents for use as steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.

miR-122 targeting with LNA/2′-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA–peptide conjugates

“…The mixmer LNA/OMe ON we designed had 10/23 LNA approximately alternating with OMe residues. It was not the purpose of this study to optimize the ratio of the two analogs or their placement within mixmers, but we have shown previously that z40% LNA distributed evenly throughout an OMe oligomer showed best results in another steric block application (Arzumanov et al 2003). Apart from their lower binding strength, OMe ONs are degradable within cells (Baker et al 1997), which might also reflect their lower activity and explain why we have not observed the OMe ON in a Northern blot when probing with an anti-inhibitor ON, in contrast to a LNA/OMe-treated sample, where it is clearly visible (Supplemental Fig.…”

Potent and sustained cellular inhibition of miR-122 by lysine-derivatized peptide nucleic acids (PNA) and phosphorothioate locked nucleic acid (LNA)/2'-O-methyl (OMe) mixmer anti-miRs in the absence of transfection agents

“…2B). We have for several years advocated the use of mixmers of LNA and OMe as steric blocking agents, 15,[46][47][48] since the binding strength of OMe residues is higher than that of 2'-deoxynucleotides, because both OMe and LNA residues intrinsically adopt a C-3'-endo, ribose-like conformation. Since the LNA content of the LNA/DNA PO ON is not revealed by the supplier, we cannot rule out the possibility that the increased potency is partly due to a significantly different LNA content between the two ONs, but it seems likely that the commercial LNA/DNA PO probe will have a similar LNA content to the LNA/OMe PO anti-miR (40% LNA).…”

Locked Nucleic Acid: High-Affinity Targeting of Complementary RNA for RNomics

“…Effects of different LNAs on the interactions of the human immunodeficiency virus (HIV)-1 trans-activation responsive element (TAR) have recently been published (Arzumanov et al 2003). Binding of oligonucleotides to TAR can inhibit Tat-dependent transcription thereby blocking full-length HIV transcription and hence viral replication, and chimeric sequences composed of LNA and 2 -O-methyl RNA nucleotides were shown to inhibit transcription in vitro (Arzumanov et al 2003).…”

“…Effects of different LNAs on the interactions of the human immunodeficiency virus (HIV)-1 trans-activation responsive element (TAR) have recently been published (Arzumanov et al 2003). Binding of oligonucleotides to TAR can inhibit Tat-dependent transcription thereby blocking full-length HIV transcription and hence viral replication, and chimeric sequences composed of LNA and 2 -O-methyl RNA nucleotides were shown to inhibit transcription in vitro (Arzumanov et al 2003). Various LNA oligonucleotides were transfected into HeLa cells and derivatives with a minimum length of 12 residues showed 50% inhibition using nanomolar concentration of LNAs (Arzumanov et al 2003), revealing the potential of LNA antisense oligonucleotides for in vivo targeting of RNA using non-RNase H dependent approaches.…”