Cellular uptake of 2‐aminopyridine‐modified peptide nucleic acids conjugated with cell‐penetrating peptides

Abstract

Cell‐penetrating peptides (CPPs) have been extensively used to deliver peptide nucleic acid (PNA) in cells. We have previously found that replacement of cytosine in triplex‐forming PNAs with 2‐aminopyridine (M) not only enhanced RNA binding, but also improved cellular uptake of PNAs. In this study, we used confocal fluorescence microscopy to evaluate the ability of CPPs to further improve cellular uptake of M‐modified PNAs. We found that PNAs conjugated with Tat and octa‐arginine peptides were effectively taken up in MCF7 cells when supplied in cell media at 1 μM. Remarkably, M‐modified PNA without any CPP conjugation also showed strong uptake when the concentration was increased to 5 μM. Majority of PNA conjugates remained localized in distinct cytoplasmic vesicles, as judged by dot‐like fluorescence patterns. However, M‐modified PNAs conjugated with Tat, octa‐arginine, and even a simple tri‐lysine peptide also showed dispersed fluorescence in cytoplasm and were taken up in nuclei where they localized in larger vesicles, most likely nucleoli. Endosomolytic peptides or chemicals (chloroquine and CaCl2) did not release the conjugates from cytosolic vesicles, which suggested that the PNAs were not entrapped in endosomes. We hypothesize that M‐modified PNAs escape endosomes and accumulate in cellular compartments rich in RNA, such as nucleoli, stress granules, and P‐bodies.

Thiazole Containing PNA Mimic Regulates c-MYC Gene Expression through DNA G-Quadruplex

“…Thus, substituting the nucleic acid bases with identical aminothiazole moieties enabled cellular uptake of the PNA like Thz-3 ligand. Such observations are consistent with previous reports (only a few) on enhanced cellular permeability of base-modified PNAs compared to unmodified ones. ,, …”

“…Such observations are consistent with previous reports (only a few) on enhanced cellular permeability of base-modified PNAs compared to unmodified ones. 18,45,46 Thz-3 Mediated Downregulation of c-MYC Expression. The transcriptional arrest by ligands Thz-2 and Thz-3 was investigated in HeLa cells (Figures 3b and S20).…”

“…For instance, introduction of 2-aminopyridine as the nucleobase in triplex-forming PNAs or incorporation of guanidine groups to the PNA backbone led to increased stability as well as cellular permeation . It has been shown that conjugation of cell-penetrating peptides like Tat peptides or addition of arginine or lysine side chains to PNAs stimulated their cellular permeation like naturally occurring proteins . However, there is evidence of accumulation of peptide conjugated PNAs in the endosomes resulting in decreased functional activities …”

Comparison of 2-Aminopyridine and 4-Thiopseudisocytosine PNA Nucleobases for Hoogsteen Recognition of Guanosine in RNA

“…Similar to L, M did not form Watson–Crick base pairs with any of the natural nucleobases minimizing off-target binding to single stranded nucleic acids . Moreover, the partial charge improves cellular uptake of M-modified PNAs, most likely due to resemblance of arginine structure that stimulates endocytosis and may be enhancing endosomal escape through a proton sponge mechanism . M-modified triplex forming PNAs are easy to synthesize and have already been used by us and others to direct RNA-specific templated reactions, suppress mRNA translation, inhibit microRNA maturation, and control conformation of dynamic structures of RNA bulges .…”

“…21 Moreover, the partial charge improves cellular uptake of M-modified PNAs, 22 most likely due to resemblance of arginine structure that stimulates endocytosis and may be enhancing endosomal escape through a proton sponge mechanism. 23 M-modified triplex forming PNAs are easy to synthesize 24 and have already been used by us and others to direct RNA-specific templated reactions, 25 suppress mRNA translation, 26 inhibit microRNA maturation, 27 and control conformation of dynamic structures of RNA bulges. 28 In our hands, M has become the most impactful PNA modification that both enables strong and selective binding to dsRNA and improves the cellular uptake of triplex-forming PNAs.…”