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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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Related in: MedlinePlus

(A) Effect of 100 µM chloroquine or 6 mM CaCl2 on Cys-K-PNA23mer-K3 anti-miR activity as seen by luciferase assay. Huh7 cells were incubated with the indicated amounts of PNA in the presence or absence of chloroquine or CaCl2. (B) Effect of CaCl2 on Cys-K-PNA23mer-K3 anti-miR activity due to increase of endocytotic uptake or enhanced endosomal escape (see text for details) as seen by luciferase assay.
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gkr885-F5: (A) Effect of 100 µM chloroquine or 6 mM CaCl2 on Cys-K-PNA23mer-K3 anti-miR activity as seen by luciferase assay. Huh7 cells were incubated with the indicated amounts of PNA in the presence or absence of chloroquine or CaCl2. (B) Effect of CaCl2 on Cys-K-PNA23mer-K3 anti-miR activity due to increase of endocytotic uptake or enhanced endosomal escape (see text for details) as seen by luciferase assay.

Mentions: Huh7 cells expressing miR-122 dual luciferase reporter construct were incubated for 4 h with 1 or 5 µM Cys-K-PNA23mer-K3 in 100 µl opti-MEM in the presence or absence of 6 mM CaCl2 or 100 µM chloroquine. After PNA incubation media was replaced by Full Media. Luciferase expression was carried out 48 h after PNA incubation. For Figure 5B, PNA-treated cells were washed twice with 150 µl PBS and media was replaced by 100 µl opti-MEM in the presence or absence of 6 mM CaCl2 but without PNA present. Four hours later, cells were washed and media was replaced by Full Media and Luciferase expression measured after 48 h.


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 100 µM chloroquine or 6 mM CaCl2 on Cys-K-PNA23mer-K3 anti-miR activity as seen by luciferase assay. Huh7 cells were incubated with the indicated amounts of PNA in the presence or absence of chloroquine or CaCl2. (B) Effect of CaCl2 on Cys-K-PNA23mer-K3 anti-miR activity due to increase of endocytotic uptake or enhanced endosomal escape (see text for details) as seen by luciferase assay.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr885-F5: (A) Effect of 100 µM chloroquine or 6 mM CaCl2 on Cys-K-PNA23mer-K3 anti-miR activity as seen by luciferase assay. Huh7 cells were incubated with the indicated amounts of PNA in the presence or absence of chloroquine or CaCl2. (B) Effect of CaCl2 on Cys-K-PNA23mer-K3 anti-miR activity due to increase of endocytotic uptake or enhanced endosomal escape (see text for details) as seen by luciferase assay.
Mentions: Huh7 cells expressing miR-122 dual luciferase reporter construct were incubated for 4 h with 1 or 5 µM Cys-K-PNA23mer-K3 in 100 µl opti-MEM in the presence or absence of 6 mM CaCl2 or 100 µM chloroquine. After PNA incubation media was replaced by Full Media. Luciferase expression was carried out 48 h after PNA incubation. For Figure 5B, PNA-treated cells were washed twice with 150 µl PBS and media was replaced by 100 µl opti-MEM in the presence or absence of 6 mM CaCl2 but without PNA present. Four hours later, cells were washed and media was replaced by Full Media and Luciferase expression measured after 48 h.

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
Related in: MedlinePlus