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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) Representative cell fractionation experiment: protein analysis by western blot showing enrichment of markers for membrane-bound compartments in the ‘pellet’ fraction as compared to the ‘supernatant’ (cytosolic) fraction. Cys-K-(TO)PNA-K3 was detected by north-western blot approach (same gel as western Blot). RNA analysis for miR-122 detection by northern blot (same samples as protein gels shown above). (B)Representative experiment of immuno-precipitation for Syntaxin 13 (STX13)-positive compartments. Top panel: western blot and north-western blot for detection of endosomal markers and Cys-K-(TO)PNA-K3, respectively. Input: ~20% IP. Bottom panel: RT–qPCR for miR-122 detection in RNA extracted from samples treated as in B top panel. Input: ~65% IP. Ag: STX13 antigen. TX-100: TritonX-100 elution (mild detergent). pH: pH shock elution.
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gkr885-F7: (A) Representative cell fractionation experiment: protein analysis by western blot showing enrichment of markers for membrane-bound compartments in the ‘pellet’ fraction as compared to the ‘supernatant’ (cytosolic) fraction. Cys-K-(TO)PNA-K3 was detected by north-western blot approach (same gel as western Blot). RNA analysis for miR-122 detection by northern blot (same samples as protein gels shown above). (B)Representative experiment of immuno-precipitation for Syntaxin 13 (STX13)-positive compartments. Top panel: western blot and north-western blot for detection of endosomal markers and Cys-K-(TO)PNA-K3, respectively. Input: ~20% IP. Bottom panel: RT–qPCR for miR-122 detection in RNA extracted from samples treated as in B top panel. Input: ~65% IP. Ag: STX13 antigen. TX-100: TritonX-100 elution (mild detergent). pH: pH shock elution.

Mentions: Cell fractionation and immunoprecipitation (IP) experiments were based on Steuble et al. (38) with some modifications (see Supplementary Methods). For Figure 7A, the membrane fraction was resuspended in 12 ml HB, while for IP (Figure 7B) it was suspended in 3.5 ml HB. For STX13 plus antigen IP experiment (Figure 7B, lane 5), beads were incubated with 5 µg pure STX13 protein (Synaptic Systems; 110-13 P) in 500 µl final volume HB for 30 min at 4°C prior to incubation with membrane fraction sample.


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) Representative cell fractionation experiment: protein analysis by western blot showing enrichment of markers for membrane-bound compartments in the ‘pellet’ fraction as compared to the ‘supernatant’ (cytosolic) fraction. Cys-K-(TO)PNA-K3 was detected by north-western blot approach (same gel as western Blot). RNA analysis for miR-122 detection by northern blot (same samples as protein gels shown above). (B)Representative experiment of immuno-precipitation for Syntaxin 13 (STX13)-positive compartments. Top panel: western blot and north-western blot for detection of endosomal markers and Cys-K-(TO)PNA-K3, respectively. Input: ~20% IP. Bottom panel: RT–qPCR for miR-122 detection in RNA extracted from samples treated as in B top panel. Input: ~65% IP. Ag: STX13 antigen. TX-100: TritonX-100 elution (mild detergent). pH: pH shock elution.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr885-F7: (A) Representative cell fractionation experiment: protein analysis by western blot showing enrichment of markers for membrane-bound compartments in the ‘pellet’ fraction as compared to the ‘supernatant’ (cytosolic) fraction. Cys-K-(TO)PNA-K3 was detected by north-western blot approach (same gel as western Blot). RNA analysis for miR-122 detection by northern blot (same samples as protein gels shown above). (B)Representative experiment of immuno-precipitation for Syntaxin 13 (STX13)-positive compartments. Top panel: western blot and north-western blot for detection of endosomal markers and Cys-K-(TO)PNA-K3, respectively. Input: ~20% IP. Bottom panel: RT–qPCR for miR-122 detection in RNA extracted from samples treated as in B top panel. Input: ~65% IP. Ag: STX13 antigen. TX-100: TritonX-100 elution (mild detergent). pH: pH shock elution.
Mentions: Cell fractionation and immunoprecipitation (IP) experiments were based on Steuble et al. (38) with some modifications (see Supplementary Methods). For Figure 7A, the membrane fraction was resuspended in 12 ml HB, while for IP (Figure 7B) it was suspended in 3.5 ml HB. For STX13 plus antigen IP experiment (Figure 7B, lane 5), beads were incubated with 5 µg pure STX13 protein (Synaptic Systems; 110-13 P) in 500 µl final volume HB for 30 min at 4°C prior to incubation with membrane fraction sample.

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