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Cellular antisense activity of peptide nucleic acid (PNAs) targeted to HIV-1 polypurine tract (PPT) containing RNA.

Boutimah-Hamoudi F, Leforestier E, Sénamaud-Beaufort C, Nielsen PE, Giovannangeli C, Saison-Behmoaras TE - Nucleic Acids Res. (2007)

Bottom Line: Our study shows that the (UUAAAAGAAAAGGGGGGAU) RNA sequence, from the human immunodeficiency virus type 1 (HIV-1 polypurine tract or PPT sequence) forms in vitro a stable folded structure involving the G-run.We have investigated the ability of pyrimidine peptide nucleic acid (PNA) oligomers targeted to the PPT sequence to invade the folded RNA and exhibit biological activity at the translation level in vitro and in cells.Interestingly, we find that both C-rich and T-rich PNAs arrested in vitro translation elongation specifically at the PPT target site.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U565, Acides nucléiques: dynamique, ciblage et fonctions biologiques, 57 rue Cuvier, CP26, Paris Cedex 05, F-75231, France.

ABSTRACT
DNA and RNA oligomers that contain stretches of guanines can associate to form stable secondary structures including G-quadruplexes. Our study shows that the (UUAAAAGAAAAGGGGGGAU) RNA sequence, from the human immunodeficiency virus type 1 (HIV-1 polypurine tract or PPT sequence) forms in vitro a stable folded structure involving the G-run. We have investigated the ability of pyrimidine peptide nucleic acid (PNA) oligomers targeted to the PPT sequence to invade the folded RNA and exhibit biological activity at the translation level in vitro and in cells. We find that PNAs can form stable complexes even with the structured PPT RNA target at neutral pH. We show that T-rich PNAs, namely the tridecamer-I PNA (C4T4CT4) forms triplex structures whereas the C-rich tridecamer-II PNA (TC6T4CT) likely forms a duplex with the target RNA. Interestingly, we find that both C-rich and T-rich PNAs arrested in vitro translation elongation specifically at the PPT target site. Finally, we show that T-rich and C-rich tridecamer PNAs that have been identified as efficient and specific blockers of translation elongation in vitro, specifically inhibit translation in streptolysin-O permeabilized cells where the PPT target sequence has been introduced upstream the reporter luciferase gene.

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Melting profiles of the complexes formed with different PNAs (4 μM) as indicated above the melting curves and complementary RNAs (RNA-I-wt, RNA-II-wt) (2 μM). Samples were prepared in a buffer containing 100 mM KCl and 10 mM sodium cacodylate, pH 7. Cooling and heating profiles are not superimposed except for complexes formed with PNA 13-mer-II. Cooling and heating rates of 0.5°C/min were used in these experiments.
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Figure 4: Melting profiles of the complexes formed with different PNAs (4 μM) as indicated above the melting curves and complementary RNAs (RNA-I-wt, RNA-II-wt) (2 μM). Samples were prepared in a buffer containing 100 mM KCl and 10 mM sodium cacodylate, pH 7. Cooling and heating profiles are not superimposed except for complexes formed with PNA 13-mer-II. Cooling and heating rates of 0.5°C/min were used in these experiments.

Mentions: UV-melting curves were performed to further characterize the different complexes. For the two PNA tridecamers, 13-mer-I and 13-mer-II, the melting profiles are very different (Figure 4); the heating and cooling curves are superimposed for the 13-mer-II in contrast to that is observed for the 13-mer-I. In the latter case, temperatures of dissociation and association depend on the heating rate, supporting non-equilibrium conditions during experiment which gave rise to hysteresis phenomenon. This observation is consistent with duplex formation with 13-mer-II PNA and triplex formation with 13-mer-I PNA since triplex formation is known to be slower than duplex one (23). At acidic pH the melting curves of 13-mer-II PNA were similar to those obtained at neutral pH, suggesting that cytosine protonation is not involved in complex stability as expected for triplexes formed with C-rich third strand (data not shown). Altogether, these results suggest that tridecamer PNAs form at neutral pH stable complexes involving duplex (13-mer-II) or triplex (13-mer-I). To better characterize the complex formed with the 13-mer-I PNA on the PPT RNA, melting profiles were performed with the 9-mer PNA that forms a triplex on the A4GA4 sequence. In the models proposed for triplex formation with T-rich PNAs, Tass reflects the non-equilibrium formation of triplex while Tdis reflects in most cases the duplex dissociation (24). Our experiments are consistent with this model; Tdis values were almost the same (Tdiss (9-mer) = 84°C and Tdis (13-mer-I) = 88°C) but Tass are completely different (Tass (9-mer) = 58°C and Tass (13-mer-I) = 70°C).Figure 4.


Cellular antisense activity of peptide nucleic acid (PNAs) targeted to HIV-1 polypurine tract (PPT) containing RNA.

Boutimah-Hamoudi F, Leforestier E, Sénamaud-Beaufort C, Nielsen PE, Giovannangeli C, Saison-Behmoaras TE - Nucleic Acids Res. (2007)

Melting profiles of the complexes formed with different PNAs (4 μM) as indicated above the melting curves and complementary RNAs (RNA-I-wt, RNA-II-wt) (2 μM). Samples were prepared in a buffer containing 100 mM KCl and 10 mM sodium cacodylate, pH 7. Cooling and heating profiles are not superimposed except for complexes formed with PNA 13-mer-II. Cooling and heating rates of 0.5°C/min were used in these experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919497&req=5

Figure 4: Melting profiles of the complexes formed with different PNAs (4 μM) as indicated above the melting curves and complementary RNAs (RNA-I-wt, RNA-II-wt) (2 μM). Samples were prepared in a buffer containing 100 mM KCl and 10 mM sodium cacodylate, pH 7. Cooling and heating profiles are not superimposed except for complexes formed with PNA 13-mer-II. Cooling and heating rates of 0.5°C/min were used in these experiments.
Mentions: UV-melting curves were performed to further characterize the different complexes. For the two PNA tridecamers, 13-mer-I and 13-mer-II, the melting profiles are very different (Figure 4); the heating and cooling curves are superimposed for the 13-mer-II in contrast to that is observed for the 13-mer-I. In the latter case, temperatures of dissociation and association depend on the heating rate, supporting non-equilibrium conditions during experiment which gave rise to hysteresis phenomenon. This observation is consistent with duplex formation with 13-mer-II PNA and triplex formation with 13-mer-I PNA since triplex formation is known to be slower than duplex one (23). At acidic pH the melting curves of 13-mer-II PNA were similar to those obtained at neutral pH, suggesting that cytosine protonation is not involved in complex stability as expected for triplexes formed with C-rich third strand (data not shown). Altogether, these results suggest that tridecamer PNAs form at neutral pH stable complexes involving duplex (13-mer-II) or triplex (13-mer-I). To better characterize the complex formed with the 13-mer-I PNA on the PPT RNA, melting profiles were performed with the 9-mer PNA that forms a triplex on the A4GA4 sequence. In the models proposed for triplex formation with T-rich PNAs, Tass reflects the non-equilibrium formation of triplex while Tdis reflects in most cases the duplex dissociation (24). Our experiments are consistent with this model; Tdis values were almost the same (Tdiss (9-mer) = 84°C and Tdis (13-mer-I) = 88°C) but Tass are completely different (Tass (9-mer) = 58°C and Tass (13-mer-I) = 70°C).Figure 4.

Bottom Line: Our study shows that the (UUAAAAGAAAAGGGGGGAU) RNA sequence, from the human immunodeficiency virus type 1 (HIV-1 polypurine tract or PPT sequence) forms in vitro a stable folded structure involving the G-run.We have investigated the ability of pyrimidine peptide nucleic acid (PNA) oligomers targeted to the PPT sequence to invade the folded RNA and exhibit biological activity at the translation level in vitro and in cells.Interestingly, we find that both C-rich and T-rich PNAs arrested in vitro translation elongation specifically at the PPT target site.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U565, Acides nucléiques: dynamique, ciblage et fonctions biologiques, 57 rue Cuvier, CP26, Paris Cedex 05, F-75231, France.

ABSTRACT
DNA and RNA oligomers that contain stretches of guanines can associate to form stable secondary structures including G-quadruplexes. Our study shows that the (UUAAAAGAAAAGGGGGGAU) RNA sequence, from the human immunodeficiency virus type 1 (HIV-1 polypurine tract or PPT sequence) forms in vitro a stable folded structure involving the G-run. We have investigated the ability of pyrimidine peptide nucleic acid (PNA) oligomers targeted to the PPT sequence to invade the folded RNA and exhibit biological activity at the translation level in vitro and in cells. We find that PNAs can form stable complexes even with the structured PPT RNA target at neutral pH. We show that T-rich PNAs, namely the tridecamer-I PNA (C4T4CT4) forms triplex structures whereas the C-rich tridecamer-II PNA (TC6T4CT) likely forms a duplex with the target RNA. Interestingly, we find that both C-rich and T-rich PNAs arrested in vitro translation elongation specifically at the PPT target site. Finally, we show that T-rich and C-rich tridecamer PNAs that have been identified as efficient and specific blockers of translation elongation in vitro, specifically inhibit translation in streptolysin-O permeabilized cells where the PPT target sequence has been introduced upstream the reporter luciferase gene.

Show MeSH
Related in: MedlinePlus