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We have tested the inhibitory potential of peptide nucleic acid (PNA) on in vitro reverse transcription of the HIV-1 gag gene. PNA was designed to target different regions of the HIV-1 gag gene and the effect on reverse transcription by HIV-1, MMLV and AMV reverse transcriptases (RTs) was investigated. We found that a bis-PNA (parallel antisense 10mer linked to antiparallel antisense 10mer) was superior to both the parallel antisense 10mer and antiparallel antisense 10mer in inhibiting reverse transcription of the gene, thus indicating triplex formation at the target sequence. A complete arrest of reverse transcription was obtained at approximately 6-fold molar excess of the bis-PNA with respect to the gag RNA. At this molar ratio we found no effect on in vitro translation of gag RNA. A 15mer duplex-forming PNA was also found to inhibit reverse transcription at very low molar ratios of PNA/ gag RNA. Specificity of the inhibition of reverse transcription by PNA was confirmed by RNA sequencing, which revealed that all tested RTs were stopped by the PNA/RNA complex at the predicted site. We propose that the effect of PNA is exclusively due to steric hindrance, as we found no signs of RNA degradation that would indicate PNA-mediated RNase H activation of the tested RTs. In conclusion, PNA appears to have a potential to become a specific and efficient inhibitor of reverse transcription in vivo , provided sufficient intracellular levels are achievable.

Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA