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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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The 32P-labelled RNA-III-wt and RNA-III-mut were incubated in 50 mM Tris (pH 8) buffer with increasing concentration of PNA (50, 200 and 400 nM) and loaded on a 15% polyacrylamide, 7 M urea gel. Arrows indicate the migration position of wild-type and mutated RNAs and of complexes s, f, C1 and C2 as described in Figure 2 legend.
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Figure 3: The 32P-labelled RNA-III-wt and RNA-III-mut were incubated in 50 mM Tris (pH 8) buffer with increasing concentration of PNA (50, 200 and 400 nM) and loaded on a 15% polyacrylamide, 7 M urea gel. Arrows indicate the migration position of wild-type and mutated RNAs and of complexes s, f, C1 and C2 as described in Figure 2 legend.

Mentions: In order to explore the stability of folded RNA and of its complexes with different PNAs we loaded the samples on denaturing acrylamide (7 M urea) gel. Figure 3 shows that wild-type RNA structures and complexes with PNA resist to the denaturing conditions. Complexes formed with mutated RNA are less stable in these conditions since complete shift of RNA was not achieved at the concentrations observed in non-denaturing gel.Figure 3.


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)

The 32P-labelled RNA-III-wt and RNA-III-mut were incubated in 50 mM Tris (pH 8) buffer with increasing concentration of PNA (50, 200 and 400 nM) and loaded on a 15% polyacrylamide, 7 M urea gel. Arrows indicate the migration position of wild-type and mutated RNAs and of complexes s, f, C1 and C2 as described in Figure 2 legend.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: The 32P-labelled RNA-III-wt and RNA-III-mut were incubated in 50 mM Tris (pH 8) buffer with increasing concentration of PNA (50, 200 and 400 nM) and loaded on a 15% polyacrylamide, 7 M urea gel. Arrows indicate the migration position of wild-type and mutated RNAs and of complexes s, f, C1 and C2 as described in Figure 2 legend.
Mentions: In order to explore the stability of folded RNA and of its complexes with different PNAs we loaded the samples on denaturing acrylamide (7 M urea) gel. Figure 3 shows that wild-type RNA structures and complexes with PNA resist to the denaturing conditions. Complexes formed with mutated RNA are less stable in these conditions since complete shift of RNA was not achieved at the concentrations observed in non-denaturing gel.Figure 3.

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