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Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs.

Torres AG, Fabani MM, Vigorito E, Williams D, Al-Obaidi N, Wojciechowski F, Hudson RH, Seitz O, Gait MJ - Nucleic Acids Res. (2011)

Bottom Line: We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes.With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments.Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.

ABSTRACT
Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.

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(A) Kinetics of uptake for Cys-K-PNA23mer-K3 anti-miR. Huh7 cells were treated with 0.5 µM PNA for the indicated time. Cells were then washed and luciferase expression was measured 48 h after PNA incubation. (B) Effect of endocytosis inhibitors on Cys-K-PNA23mer-K3 uptake as seen by luciferase assay. CPZ: chlorpromazine; Lat B: Latrunculin B; MBCD: Methyl-β-cyclodextrin; AP180-C: dominant-negative form of AP180.
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gkr885-F4: (A) Kinetics of uptake for Cys-K-PNA23mer-K3 anti-miR. Huh7 cells were treated with 0.5 µM PNA for the indicated time. Cells were then washed and luciferase expression was measured 48 h after PNA incubation. (B) Effect of endocytosis inhibitors on Cys-K-PNA23mer-K3 uptake as seen by luciferase assay. CPZ: chlorpromazine; Lat B: Latrunculin B; MBCD: Methyl-β-cyclodextrin; AP180-C: dominant-negative form of AP180.

Mentions: Dual-luciferase reporter Huh7 cells were incubated with 0.5 µM Cys-K-PNA23mer-K3 (a concentration at which PNA activity is sub-saturating, Figure 1A) for various times up to 240 min in serum-free media. After PNA incubations, cells were washed thoroughly to remove undelivered PNA, media replaced by Full Media and Luciferase expression measured after 48 h as before. The activity data plotted against time fit to a sigmoidal-shaped curve (Figure 4A). A low level of activity (RLuc/FLuc 1.5–2.0) is obtained within 5 min of PNA anti-miR incubation, which is followed by a rapid increase after about 15 min and reached a plateau by 100 min incubation time. We ruled out the possibility that the low-level activity at early time-points is due to Cys-K-PNA23-mer-K3 remaining bound to the plasma membrane after washing and internalized during subsequent cell growth, because in a control experiment where cells were incubated with Cys-K-PNA23-mer-K3 for 15 min, washed thoroughly and further incubated for 1 h with excess of scrambled Cys-K-PNA23-mer-K3 control anti-miR as competitor, no decrease in Cys-K-PNA23-mer-K3 activity was observed (Supplementary Figure S2).Figure 4.


Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs.

Torres AG, Fabani MM, Vigorito E, Williams D, Al-Obaidi N, Wojciechowski F, Hudson RH, Seitz O, Gait MJ - Nucleic Acids Res. (2011)

(A) Kinetics of uptake for Cys-K-PNA23mer-K3 anti-miR. Huh7 cells were treated with 0.5 µM PNA for the indicated time. Cells were then washed and luciferase expression was measured 48 h after PNA incubation. (B) Effect of endocytosis inhibitors on Cys-K-PNA23mer-K3 uptake as seen by luciferase assay. CPZ: chlorpromazine; Lat B: Latrunculin B; MBCD: Methyl-β-cyclodextrin; AP180-C: dominant-negative form of AP180.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300011&req=5

gkr885-F4: (A) Kinetics of uptake for Cys-K-PNA23mer-K3 anti-miR. Huh7 cells were treated with 0.5 µM PNA for the indicated time. Cells were then washed and luciferase expression was measured 48 h after PNA incubation. (B) Effect of endocytosis inhibitors on Cys-K-PNA23mer-K3 uptake as seen by luciferase assay. CPZ: chlorpromazine; Lat B: Latrunculin B; MBCD: Methyl-β-cyclodextrin; AP180-C: dominant-negative form of AP180.
Mentions: Dual-luciferase reporter Huh7 cells were incubated with 0.5 µM Cys-K-PNA23mer-K3 (a concentration at which PNA activity is sub-saturating, Figure 1A) for various times up to 240 min in serum-free media. After PNA incubations, cells were washed thoroughly to remove undelivered PNA, media replaced by Full Media and Luciferase expression measured after 48 h as before. The activity data plotted against time fit to a sigmoidal-shaped curve (Figure 4A). A low level of activity (RLuc/FLuc 1.5–2.0) is obtained within 5 min of PNA anti-miR incubation, which is followed by a rapid increase after about 15 min and reached a plateau by 100 min incubation time. We ruled out the possibility that the low-level activity at early time-points is due to Cys-K-PNA23-mer-K3 remaining bound to the plasma membrane after washing and internalized during subsequent cell growth, because in a control experiment where cells were incubated with Cys-K-PNA23-mer-K3 for 15 min, washed thoroughly and further incubated for 1 h with excess of scrambled Cys-K-PNA23-mer-K3 control anti-miR as competitor, no decrease in Cys-K-PNA23-mer-K3 activity was observed (Supplementary Figure S2).Figure 4.

Bottom Line: We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes.With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments.Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.

ABSTRACT
Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.

Show MeSH