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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) Gel mobility-shift analysis of PNAs binding to their target RNA. A fixed concentration of the 32P-labeled RNA-III-wt and mismatches containing RNA-III-mut (2 × 10−9 M) were incubated in 50 mM Tris (pH 8) buffer with increasing concentration of PNA as indicated above the lanes. Slow migrating complexes (s) correspond to the complexes formed with folded RNA whereas higher electrophoretic mobility complexes (f) correspond to the invasion complexes that unfold the structured RNA target. C1 and C2 denote complexes involving one and two molecules of bis-PNAs respectively. (B) An excess of the RNA target sequence (2.5-fold excess relative to PNA concentration (400 nM)) was added to the complexes and incubated for 5 min. Bound and unbound complexes were separated in a 15% non-denaturing polyacrylamide gel at 20°C. RNA-III-wt (top) and RNA-III-mut (bottom) were used as targets.
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Figure 2: (A) Gel mobility-shift analysis of PNAs binding to their target RNA. A fixed concentration of the 32P-labeled RNA-III-wt and mismatches containing RNA-III-mut (2 × 10−9 M) were incubated in 50 mM Tris (pH 8) buffer with increasing concentration of PNA as indicated above the lanes. Slow migrating complexes (s) correspond to the complexes formed with folded RNA whereas higher electrophoretic mobility complexes (f) correspond to the invasion complexes that unfold the structured RNA target. C1 and C2 denote complexes involving one and two molecules of bis-PNAs respectively. (B) An excess of the RNA target sequence (2.5-fold excess relative to PNA concentration (400 nM)) was added to the complexes and incubated for 5 min. Bound and unbound complexes were separated in a 15% non-denaturing polyacrylamide gel at 20°C. RNA-III-wt (top) and RNA-III-mut (bottom) were used as targets.

Mentions: In order to determine whether short complementary PNAs can invade and bind to structured RNA targets, here RNA-III-wt, we have used electrophoretic mobility shift assays. Binding of PNAs to unfolded RNA-III-mut was also analysed. 32P-radiolabelled RNAs were incubated in the absence or in the presence of increasing concentrations of tridecamer PNAs, 13-mer-I and 13-mer-II. Figure 2A shows that at low concentrations (<25 nM) tridecamers form complexes that migrate slower than folded RNA [complex (s)] while at higher concentrations a discrete, faster migrating complexes [complex (f)] were predominant. We postulated that retarded low mobility species result from the fixation of PNAs to the A-rich region of the target sequence not engaged in the structure formed by the G-tract while high mobility complexes result from the invasion of RNA structures by 13-mer-I and 13-mer-II PNAs.13-mer-II PNA that can form six C.G base pairs invades slightly more efficiently folded RNA than 13-mer-I PNA that can form only four C.G base pairs with the PPT RNA target. The binding of the two tridecamer PNAs was also studied on the mutated target, RNA-III-mut. This RNA sequence is not stably structured and is complementary on 11 and 8 contiguous nucleotides with 13-mer-I and 13-mer-II PNAs, respectively. Then, binding of 13-mer-PNAs can be also observed on this RNA sequence, with complexes migrating at the same position as the ones observed on the wild-type sequence after PNA-induced unfolding (Figure 2A). Binding efficiencies of 13-mer-PNAs on the two RNA targets, wild type and mutated, was measured. For tridecamer-PNAs complexed to RNA-III-wt, we could not determine precisely the K50 values (that means the concentrations of PNAs required for the formation of 50% of complex) because of the multiplicity of the slow-mobility complexes, however these values are situated between 2 and 5 nM. On the contrary, K50 values can be determined on unstructured RNA-III mut; for unmodified and acridine-modified tridecamer-PNAs complexed to RNA-III-mut, K50 was determined around 4 nM.Figure 2.


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) Gel mobility-shift analysis of PNAs binding to their target RNA. A fixed concentration of the 32P-labeled RNA-III-wt and mismatches containing RNA-III-mut (2 × 10−9 M) were incubated in 50 mM Tris (pH 8) buffer with increasing concentration of PNA as indicated above the lanes. Slow migrating complexes (s) correspond to the complexes formed with folded RNA whereas higher electrophoretic mobility complexes (f) correspond to the invasion complexes that unfold the structured RNA target. C1 and C2 denote complexes involving one and two molecules of bis-PNAs respectively. (B) An excess of the RNA target sequence (2.5-fold excess relative to PNA concentration (400 nM)) was added to the complexes and incubated for 5 min. Bound and unbound complexes were separated in a 15% non-denaturing polyacrylamide gel at 20°C. RNA-III-wt (top) and RNA-III-mut (bottom) were used as targets.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: (A) Gel mobility-shift analysis of PNAs binding to their target RNA. A fixed concentration of the 32P-labeled RNA-III-wt and mismatches containing RNA-III-mut (2 × 10−9 M) were incubated in 50 mM Tris (pH 8) buffer with increasing concentration of PNA as indicated above the lanes. Slow migrating complexes (s) correspond to the complexes formed with folded RNA whereas higher electrophoretic mobility complexes (f) correspond to the invasion complexes that unfold the structured RNA target. C1 and C2 denote complexes involving one and two molecules of bis-PNAs respectively. (B) An excess of the RNA target sequence (2.5-fold excess relative to PNA concentration (400 nM)) was added to the complexes and incubated for 5 min. Bound and unbound complexes were separated in a 15% non-denaturing polyacrylamide gel at 20°C. RNA-III-wt (top) and RNA-III-mut (bottom) were used as targets.
Mentions: In order to determine whether short complementary PNAs can invade and bind to structured RNA targets, here RNA-III-wt, we have used electrophoretic mobility shift assays. Binding of PNAs to unfolded RNA-III-mut was also analysed. 32P-radiolabelled RNAs were incubated in the absence or in the presence of increasing concentrations of tridecamer PNAs, 13-mer-I and 13-mer-II. Figure 2A shows that at low concentrations (<25 nM) tridecamers form complexes that migrate slower than folded RNA [complex (s)] while at higher concentrations a discrete, faster migrating complexes [complex (f)] were predominant. We postulated that retarded low mobility species result from the fixation of PNAs to the A-rich region of the target sequence not engaged in the structure formed by the G-tract while high mobility complexes result from the invasion of RNA structures by 13-mer-I and 13-mer-II PNAs.13-mer-II PNA that can form six C.G base pairs invades slightly more efficiently folded RNA than 13-mer-I PNA that can form only four C.G base pairs with the PPT RNA target. The binding of the two tridecamer PNAs was also studied on the mutated target, RNA-III-mut. This RNA sequence is not stably structured and is complementary on 11 and 8 contiguous nucleotides with 13-mer-I and 13-mer-II PNAs, respectively. Then, binding of 13-mer-PNAs can be also observed on this RNA sequence, with complexes migrating at the same position as the ones observed on the wild-type sequence after PNA-induced unfolding (Figure 2A). Binding efficiencies of 13-mer-PNAs on the two RNA targets, wild type and mutated, was measured. For tridecamer-PNAs complexed to RNA-III-wt, we could not determine precisely the K50 values (that means the concentrations of PNAs required for the formation of 50% of complex) because of the multiplicity of the slow-mobility complexes, however these values are situated between 2 and 5 nM. On the contrary, K50 values can be determined on unstructured RNA-III mut; for unmodified and acridine-modified tridecamer-PNAs complexed to RNA-III-mut, K50 was determined around 4 nM.Figure 2.

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