Limits...
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.

Show MeSH
(A) Effect of Cys modification or replacement within Cys-K-PNA23mer-K3 on anti-miR activity as seen by luciferase assay. Huh7 cells were treated with 1 µM PNAs. (B) Luciferase activity assay for 1 µM TO-PNAs as compared to a sequence-equivalent unmodified PNA anti-miR. (C) Representative FACS histogram for Huh7 (left panel) or HEK293ET (right panel) cells treated with TO-PNAs at 3 µM. (D) Median fluorescence relative to untreated cells observed for Huh7 cells and HEK293ET cells treated as in (C) by FACS analysis; shown are results of three independent experiments. (E) Confocal microscopy of Huh7 cells treated with TO-PNAs at 3 µM. Green, TO-PNAs; blue, Hoechst staining; red, Cell Mask plasma membrane staining. Scale bar corresponds to 5 µm. All three images were taken with the same confocal microscopy settings.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3300011&req=5

gkr885-F3: (A) Effect of Cys modification or replacement within Cys-K-PNA23mer-K3 on anti-miR activity as seen by luciferase assay. Huh7 cells were treated with 1 µM PNAs. (B) Luciferase activity assay for 1 µM TO-PNAs as compared to a sequence-equivalent unmodified PNA anti-miR. (C) Representative FACS histogram for Huh7 (left panel) or HEK293ET (right panel) cells treated with TO-PNAs at 3 µM. (D) Median fluorescence relative to untreated cells observed for Huh7 cells and HEK293ET cells treated as in (C) by FACS analysis; shown are results of three independent experiments. (E) Confocal microscopy of Huh7 cells treated with TO-PNAs at 3 µM. Green, TO-PNAs; blue, Hoechst staining; red, Cell Mask plasma membrane staining. Scale bar corresponds to 5 µm. All three images were taken with the same confocal microscopy settings.

Mentions: We further investigated the function of the terminal Cys residue in PNA anti-miRs and asked if the enhanced anti-miR activity is due to the presence of a free thiol group. PNA23-mer anti-miR was alkylated at the Cys residue using N-ethylmaleimide (NEM) and the anti-miR added to dual luciferase reporter Huh7 cells at 1 µM concentration for 4 h as before. Alkylation (dCys(NEM)-dK-PNA23mer-dK3) led to a dramatic loss in anti-miR activity (Figure 3A), similar to that of dK-PNA23mer-dK3 anti-miR lacking a Cys residue (Figure 2A). Similarly, replacement of the Cys by Met, which contains a methylated thiol group (Met-K-PNA23mer-K3), or by Ser (Ser-K-PNA23mer-K3) resulted in each case in a substantial reduction in anti-miR activity (Figure 3A). In contrast, activity was completely recovered when the Cys was replaced by 3-mercaptopropionic acid (MPA-K-PNA23mer-K3). These data strongly suggest that the enhanced PNA anti-miR activity for Cys-containing PNA anti-miRs is due to the presence of a terminal-free thiol group.Figure 3.


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) Effect of Cys modification or replacement within Cys-K-PNA23mer-K3 on anti-miR activity as seen by luciferase assay. Huh7 cells were treated with 1 µM PNAs. (B) Luciferase activity assay for 1 µM TO-PNAs as compared to a sequence-equivalent unmodified PNA anti-miR. (C) Representative FACS histogram for Huh7 (left panel) or HEK293ET (right panel) cells treated with TO-PNAs at 3 µM. (D) Median fluorescence relative to untreated cells observed for Huh7 cells and HEK293ET cells treated as in (C) by FACS analysis; shown are results of three independent experiments. (E) Confocal microscopy of Huh7 cells treated with TO-PNAs at 3 µM. Green, TO-PNAs; blue, Hoechst staining; red, Cell Mask plasma membrane staining. Scale bar corresponds to 5 µm. All three images were taken with the same confocal microscopy settings.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr885-F3: (A) Effect of Cys modification or replacement within Cys-K-PNA23mer-K3 on anti-miR activity as seen by luciferase assay. Huh7 cells were treated with 1 µM PNAs. (B) Luciferase activity assay for 1 µM TO-PNAs as compared to a sequence-equivalent unmodified PNA anti-miR. (C) Representative FACS histogram for Huh7 (left panel) or HEK293ET (right panel) cells treated with TO-PNAs at 3 µM. (D) Median fluorescence relative to untreated cells observed for Huh7 cells and HEK293ET cells treated as in (C) by FACS analysis; shown are results of three independent experiments. (E) Confocal microscopy of Huh7 cells treated with TO-PNAs at 3 µM. Green, TO-PNAs; blue, Hoechst staining; red, Cell Mask plasma membrane staining. Scale bar corresponds to 5 µm. All three images were taken with the same confocal microscopy settings.
Mentions: We further investigated the function of the terminal Cys residue in PNA anti-miRs and asked if the enhanced anti-miR activity is due to the presence of a free thiol group. PNA23-mer anti-miR was alkylated at the Cys residue using N-ethylmaleimide (NEM) and the anti-miR added to dual luciferase reporter Huh7 cells at 1 µM concentration for 4 h as before. Alkylation (dCys(NEM)-dK-PNA23mer-dK3) led to a dramatic loss in anti-miR activity (Figure 3A), similar to that of dK-PNA23mer-dK3 anti-miR lacking a Cys residue (Figure 2A). Similarly, replacement of the Cys by Met, which contains a methylated thiol group (Met-K-PNA23mer-K3), or by Ser (Ser-K-PNA23mer-K3) resulted in each case in a substantial reduction in anti-miR activity (Figure 3A). In contrast, activity was completely recovered when the Cys was replaced by 3-mercaptopropionic acid (MPA-K-PNA23mer-K3). These data strongly suggest that the enhanced PNA anti-miR activity for Cys-containing PNA anti-miRs is due to the presence of a terminal-free thiol group.Figure 3.

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