Influence of Pendant Chiral C^γ-(Alkylideneamino/Guanidino) Cationic Side-chains of PNA Backbone on Hybridization with Complementary DNA/RNA and Cell Permeability

Abstract

Intrinsically cationic and chiral C(γ)-substituted peptide nucleic acid (PNA) analogues have been synthesized in the form of γ(S)-ethyleneamino (eam)- and γ(S)-ethyleneguanidino (egd)-PNA with two carbon spacers from the backbone. The relative stabilization (ΔTm) of duplexes from modified cationic PNAs as compared to 2-aminoethylglycyl (aeg)-PNA is better with complementary DNA (PNA:DNA) than with complementary RNA (PNA:RNA). Inherently, PNA:RNA duplexes have higher stability than PNA:DNA duplexes, and the guanidino PNAs are superior to amino PNAs. The cationic PNAs were found to be specific toward their complementary DNA target as seen from their significantly lower binding with DNA having single base mismatch. The differential binding avidity of cationic PNAs was assessed by the displacement of DNA duplex intercalated ethidium bromide and gel electrophoresis. The live cell imaging of amino/guanidino PNAs demonstrated their ability to penetrate the cell membrane in 3T3 and MCF-7 cells, and cationic PNAs were found to be accumulated in the vicinity of the nuclear membrane in the cytoplasm. Fluorescence-activated cell sorter (FACS) analysis of cell permeability showed the efficiency to be dependent upon the nature of cationic functional group, with guanidino PNAs being better than the amino PNAs in both cell lines. The results are useful to design new biofunctional cationic PNA analogues that not only bind RNA better but also show improved cell permeability.

Incorporating a guanidine-modified cytosine base into triplex-forming PNAs for the recognition of a C-G pyrimidine–purine inversion site of an RNA duplex

“…Thus, the attached guanidine group in P3 stabilizes the PNA·RNA 2 triplex formation mainly through sequence specific Q·C-G base triple formation, but not non-specific ionic interaction as the case when the guanidine group is attached to the PNA backbone (16,36–38). …”

“…The cellular uptake of a DNA duplex-binding TFO with a 2′-OMe or 2′-AE RNA backbone containing a Q base is facilitated by electroporation (27). We reason that combining the guanidine group and the flexibility of PNA backbone may facilitate the penetration (37,38,46,48–53) of the Q modified PNAs through the cell membrane. Our preliminary confocal microscopy studies (Figure 7) show that a PNA incorporating three Q residues and labeled with Cy3 dye (Supplementary Table S1) is taken by HeLa cells without any transfection agent.…”

Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs): Synthesis and DNA Binding

“…A number of studies have focused on chiral PNAs with guanidine moieties in the side chains [ 26 ]. Good cell membrane permeability has been demonstrated for such PNA derivatives, and the binding efficiency of the γ- S -derivatives [ 22 , 27 – 28 ] was superior to that of the α- R -derivatives [ 28 ]. All of the above-mentioned studies on PNA modification have aimed at developing new PNA-based molecular tools for fundamental genome studies and broadening the sphere of the practical applications of PNAs [ 29 ].…”

Pyrrolidinyl PNA with α/β-Dipeptide Backbone: From Development to Applications

“…16 Also, preorganization resulting from a simple substitution at the γ-position in aegPNA significantly improves its binding properties. 17 Various substituents can be placed at this position to improve water solubility 18 or cell penetration 19,20 or to incorporate other functions. 21,22 Because of its straightforward access via proline derivatives having well-defined stereochemistries, the pyrrolidine ring has been extensively used as a constraint element in the design of new PNA structures ( Figure 3).…”