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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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(A) CMV (+PPT)/HeLa cell lines contain bi-directional CMV promoter that allows expression of two reporter genes [firefly luciferase (luc) and GFP]. The PPT-wt sequence (5′AAAAGAAAAGGGGGGA3′) was inserted upstream of the luc gene and a mutated version (PPT-mut, 5′AAAAGAAGGGGAGGAA3′, the four mutations are underlined) was inserted upstream of the GFP gene. (B) PNA tridecamers are efficient inhibitors of translation. Cells were reversibly permeabilized by SLO in the presence of various PNAs (see ‘Materials and Methods’ section). PNA concentrations in final culturing conditions are indicated.
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Figure 6: (A) CMV (+PPT)/HeLa cell lines contain bi-directional CMV promoter that allows expression of two reporter genes [firefly luciferase (luc) and GFP]. The PPT-wt sequence (5′AAAAGAAAAGGGGGGA3′) was inserted upstream of the luc gene and a mutated version (PPT-mut, 5′AAAAGAAGGGGAGGAA3′, the four mutations are underlined) was inserted upstream of the GFP gene. (B) PNA tridecamers are efficient inhibitors of translation. Cells were reversibly permeabilized by SLO in the presence of various PNAs (see ‘Materials and Methods’ section). PNA concentrations in final culturing conditions are indicated.

Mentions: SLO (Institute of Medical Microbiology and Hygiene, Mainz, Germany) was used to reversibly permeabilize CMV/(+)PPT/HeLa or CMV-luc/HeLa cell lines toward PNAs according to a recently revised protocol (17–18). SLO was conserved in PBS buffer supplemented with 0.1% BSA. Cells were washed twice and re-suspended in HBSS (Hanks’ balanced salt solution with calcium and magnesium, 10 mM HEPES, and 1% fetal bovine serum). For each experiment, in a 48-well dishes, 1.3 × 105 cells in 100 µl were permeabilized by addition of an optimized amount of SLO (110 ng) and then incubated at 37°C for 15 min, in the presence of PNAs. Resealing was achieved by addition of 800 µl of DMEM supplemented with 10% fetal calf serum and further incubation at 37°C for 20 min. Cells were then transferred in 96-well dishes (4 × 104 cells/well) and cultured at 37°C for 4 h and then induced by addition of doxycycline (Sigma) and further cultured 40 h before quantifying firefly luciferase activity, GFP and total proteins. In the same time, cells were examined with respect to permeabilization efficiency and viability by flow cytometry (Facsort, Beckton Dickinson). Briefly, during permeabilization PNAs were replaced with FITC (20 µM) and after resealing cells were washed twice with PBS and passed through flow cytometer in PBS supplemented with propidium iodide (10 µg/ml) which permitted to determine the fraction of permeabilized and viable cells in the original culture. At the end of experiment, cells were harvested for lysate (passive lysis buffer, Promega) and both the firefly luciferase activity [expressed in relative light units (RLU)], GFP fluorescence and protein concentration [Bradford reagent from Bio-Rad, expressed in optical density (OD)] were measured using a spectrofluorimeter (Wallac Victor 2 Multi-label Counter, Perkin Elmer). The luciferase activity and GFP expression shown in Figure 6 were normalized to the absorbance data, that reflect the amount of proteins, and then expressed as a percentage compared with the luciferase activity and GFP fluorescence levels that were obtained in SLO-treated cells in the absence of PNA. Each data point was averaged over two replicates of three separate experiments.


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)

(A) CMV (+PPT)/HeLa cell lines contain bi-directional CMV promoter that allows expression of two reporter genes [firefly luciferase (luc) and GFP]. The PPT-wt sequence (5′AAAAGAAAAGGGGGGA3′) was inserted upstream of the luc gene and a mutated version (PPT-mut, 5′AAAAGAAGGGGAGGAA3′, the four mutations are underlined) was inserted upstream of the GFP gene. (B) PNA tridecamers are efficient inhibitors of translation. Cells were reversibly permeabilized by SLO in the presence of various PNAs (see ‘Materials and Methods’ section). PNA concentrations in final culturing conditions are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: (A) CMV (+PPT)/HeLa cell lines contain bi-directional CMV promoter that allows expression of two reporter genes [firefly luciferase (luc) and GFP]. The PPT-wt sequence (5′AAAAGAAAAGGGGGGA3′) was inserted upstream of the luc gene and a mutated version (PPT-mut, 5′AAAAGAAGGGGAGGAA3′, the four mutations are underlined) was inserted upstream of the GFP gene. (B) PNA tridecamers are efficient inhibitors of translation. Cells were reversibly permeabilized by SLO in the presence of various PNAs (see ‘Materials and Methods’ section). PNA concentrations in final culturing conditions are indicated.
Mentions: SLO (Institute of Medical Microbiology and Hygiene, Mainz, Germany) was used to reversibly permeabilize CMV/(+)PPT/HeLa or CMV-luc/HeLa cell lines toward PNAs according to a recently revised protocol (17–18). SLO was conserved in PBS buffer supplemented with 0.1% BSA. Cells were washed twice and re-suspended in HBSS (Hanks’ balanced salt solution with calcium and magnesium, 10 mM HEPES, and 1% fetal bovine serum). For each experiment, in a 48-well dishes, 1.3 × 105 cells in 100 µl were permeabilized by addition of an optimized amount of SLO (110 ng) and then incubated at 37°C for 15 min, in the presence of PNAs. Resealing was achieved by addition of 800 µl of DMEM supplemented with 10% fetal calf serum and further incubation at 37°C for 20 min. Cells were then transferred in 96-well dishes (4 × 104 cells/well) and cultured at 37°C for 4 h and then induced by addition of doxycycline (Sigma) and further cultured 40 h before quantifying firefly luciferase activity, GFP and total proteins. In the same time, cells were examined with respect to permeabilization efficiency and viability by flow cytometry (Facsort, Beckton Dickinson). Briefly, during permeabilization PNAs were replaced with FITC (20 µM) and after resealing cells were washed twice with PBS and passed through flow cytometer in PBS supplemented with propidium iodide (10 µg/ml) which permitted to determine the fraction of permeabilized and viable cells in the original culture. At the end of experiment, cells were harvested for lysate (passive lysis buffer, Promega) and both the firefly luciferase activity [expressed in relative light units (RLU)], GFP fluorescence and protein concentration [Bradford reagent from Bio-Rad, expressed in optical density (OD)] were measured using a spectrofluorimeter (Wallac Victor 2 Multi-label Counter, Perkin Elmer). The luciferase activity and GFP expression shown in Figure 6 were normalized to the absorbance data, that reflect the amount of proteins, and then expressed as a percentage compared with the luciferase activity and GFP fluorescence levels that were obtained in SLO-treated cells in the absence of PNA. Each data point was averaged over two replicates of three separate experiments.

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