Membrane-based hybridization capture of intracellular peptide nucleic acid

Shin, Duckhyang; Nam, Minwoo; Yoon, Yeup; Kim, Meehyein

doi:10.1016/j.ab.2009.11.031

Cited by 3 publications

(3 citation statements)

References 9 publications

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“…Cys-K-(TO)PNA-K3 was also detected in the ‘pellet’ fraction as seen by the north-western blotting approach. Note that Cys-K-(TO)PNA-K3 was detected as two bands instead of one, similarly to the reported detection of a PNA ON conjugated to a cationic cell penetrating peptide, whereas a charge neutral PNA ON of the same sequence was detected as a single band (59). Finally, northern blotting showed enrichment of mature miR-122 in the ‘pellet’ fraction as compared to the ‘supernatant’, in agreement with mature miRNAs being found within endosomal compartments (55).…”

Section: Resultssupporting

confidence: 83%

“…Proteins and RNA were extracted from each fraction and analyzed by western blot and northern blot, respectively. PNA ONs may be recovered by standard protein extraction and detected by electrophoresis as discrete bands in standard protein gels after western blotting by use of a radiolabeled RNA complementary probe (north-western blot), similarly to a previous report (59). In western blotting, there was enrichment in the ‘pellet’ fraction of markers for membrane-bound compartments including Rab5 (early endosomes), Syntaxin13 (STX13; recycling endosomes), Lysosomal-associated membrane protein 1 (LAMP1; late endosomes/lysosomes) and Golgin (Golgi apparatus) (Figure 7A).…”

Section: Resultsmentioning

confidence: 88%

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

Torres

Fabani

Vigorito

et al. 2011

Nucleic Acids Research

104

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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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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 88%

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

Torres

Fabani

Vigorito

et al. 2011

Nucleic Acids Research

104

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“…Homer1 proteins have been suggested to play critical roles in regulating synapse development, synaptic strength, and homeostatic synaptic scaling (33)(34)(35)(36)(37). At the postsynaptic density, the activity-dependent short protein isoform of the gene, Homer1a, has dominant-negative effects on the constitutive longer forms of Homer1 (Homer1b/c), and through this interaction regulates the clustering of mGluRs (34)(35)(36) and mGluRdependent NMDA and AMPA receptor currents (34,(38)(39)(40)(41). It is intriguing to theorize that circHomer1 could have a similar dominant negative affect as its linear, activitydependent counterpart, and circHomer1 may regulate synaptic Homer1b/c via such a mechanism (as discussed below(42)).…”

Section: Discussionmentioning

confidence: 99%

The noncoding circular RNAcircHomer1regulates developmental experience-dependent plasticity in mouse visual cortex

Jenks,

Cai,

Nayan

et al. 2024

Preprint

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Circular RNAs (circRNAs) are noncoding RNAs abundant in brain tissue, and many are derived from activity-dependent, linear mRNAs encoding for synaptic proteins, suggesting that circRNAs may directly or indirectly play a role in regulating synaptic development, plasticity, and function. However, it is unclear if the circular forms of these RNAs are similarly regulated by activity and what role these circRNAs play in developmental plasticity. Here, we employed transcriptome-wide analysis comparing differential expression of both mRNAs and circRNAs in juvenile mouse primary visual cortex (V1) following monocular deprivation (MD), a model of developmental plasticity. Among the differentially expressed mRNAs and circRNAs following 3-day MD, the circular and the activity-dependent linear forms of the Homer1 gene, circHomer1 and Homer1a respectively, were of interest as their expression changed in opposite directions: circHomer1 expression increased while the expression of Homer1a decreased following MD. Knockdown of circHomer1 prevented the depression of closed-eye responses normally observed after 3-day MD. circHomer1-knockdown led to a reduction in average dendritic spine size prior to MD, but critically there was no further reduction after 3-day MD, consistent with impaired structural plasticity. circHomer1-knockdown also prevented the reduction of surface AMPA receptors after 3-day MD. Synapse-localized puncta of the AMPA receptor endocytic protein Arc increased in volume after MD but were smaller in circHomer1-knockdown neurons, suggesting that circHomer1 regulates plasticity through mechanisms of activity-dependent AMPA receptor endocytosis. Thus, activity-dependent circRNAs regulate developmental synaptic plasticity, and our findings highlight the essential role of circHomer1 in V1 plasticity induced by short-term MD.

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