Modified (PNA, 2'-O-methyl and phosphoramidate) anti-TAR antisense oligonucleotides as strong and specific inhibitors of in vitro HIV-1 reverse transcription

Abstract

Natural beta-phosphodiester 16mer and 15mer antisense oligonucleotides targeted against the HIV-1 and HIV-2 TAR RNAs respectively were previously described as sequence-specific inhibitors of in vitro retroviral reverse transcription. In this work, we tested chemically modified oligonucleotide analogues: alpha-phosphodiester, phosphorothioate, methylphosphonate, peptide nucleic acid or PNA, 2'- o -methyl and (N3'-P5') phosphoramidate versions of the 16mer anti-TAR oligonucleotide. PNA, 2'- O -methyl and (N3'-P5') phosphoramidate oligomers showed a strong inhibitory effect compared with the unmodified 16mer, with reverse transcription inhibition (IC50) values in the nanomolar range. The inhibition was sequence-specific, as scrambled and mismatched control oligonucleotides were not able to inhibit cDNA synthesis. No direct binding of the 2'- O -methyl, PNA or (N3'-P5') phosphoramidate anti-TAR oligonucleotides to the HIV-1 reverse transcriptase was observed. The higher T m obtained with 2'- O -methyl, (N3'-P5') phosphoramidate and PNA molecules concerning the annealing with the stem-loop structure of the TAR RNA, in comparison with the beta-phosphodiester oligonucleotides, is correlated with their high inhibitory effect on reverse transcription.

Inhibition of HIV-1 Replication by Oligonucleotide Analogues Directed to the Packaging Signal and Trans-Activating Response Region

“…The level of virus release from Jurkat cells was dependent on the success of several stages of the virus life cycle. The smaller effect seen by the TARtargeted ON was possibly due to only viral transcription being inhibited, although this ON would be expected to also have an effect at the reverse transcription step (Boulme et al, 1998;Boulme et al, 1997). The larger inhibition of virus release seen by both chemistries of the SL3-targeted ONs might be due to multiple steps being affected, including transcription, translation and splicing, as well as viral packaging, and possibly reverse transcription.…”

“…This is present at the 5′-end of all viral transcripts. Steric block 2′-O-methyl (2′Ome; Arzumanov et al, 2001b;Boulme et al, 1998), 2′Ome/locked nucleic acid (LNA) mixmer (Arzumanov et al, 2001b), 2′ Ome G-clamp and peptide nucleic acid (Arzumanov et al, 2001b;Kaushik et al, 2002). ONs targeted to TAR displayed efficient steric block of the transactivator (Tat)-TAR interaction in vitro These ONs also inhibited Tat-dependent transcription from the viral long terminal repeats, and a 2′Ome/LNA ON had high-level activity in a HeLa-cell-based reporter assay of Tat-dependent trans-activation (Arzumanov et al, 2001b).…”

Fluorine‐18 labeling of peptide nucleic acids

“…They can be designed with a variety of chemical modifications 3 or conjugated with carriers 4 and targeting agents, 5 and hold great promise in an array of applications (reviewed in ref. 2), including several with therapeutic potential such as antisense-, 6 antigene-, 7 antitumoral- 8 and antibacterial 5 agents. The therapeutic potential of PNAs deserves to be evaluated on the basis of their in vivo behavior with adapted techniques.…”

Use of the Fluorescent Nucleoside Analogue Benzo[g]quinazoline 2′-O-Methyl--D-ribofuranoside to Monitor the Binding of the HIV-1 Tat Protein or of Antisense Oligonucleotides to the TAR RNA Stem-Loop

“…We showed recently that nuclease-stabilized 16-mer 2'-O-methyloligoribonucleotides are effective agents in steric blocking of Tat-TAR [23] [24]. The 2'-O-methyloligoribonucleotides targeted to TAR are also strong inhibitors of HIV reverse transcription [25]. Further, DNA aptamers and their 2'-O-methyl analogues have been selected to bind strongly to TAR and form kissing complexes [26].…”