Multiple Charged Purine (MCP) PNA as a Simple Mode for Cellular Uptake

Peptide nucleic acid (PNA) is a synthetic DNA analogue characterized by exceptional biostability and strong hybridization affinity toward complementary DNA and RNA. However, its inherently low membrane permeability hampers its biomedical applicability. N-Methylation of guanine and adenine PNA monomers produces a positively charged nucleobase, which suppresses the formation of self-duplexes while improving DNA affinity through electrostatic interactions. To overcome the cellular delivery limitations of PNA, we designed and synthesized a 16-mer, nontargeted model PNA incorporating 4, 6, or 8 positively charged purines (G+ and A+). As comparative controls, the corresponding unmodified PNA was conjugated to a short cell-penetrating peptide (CPP) containing 4, 6, or 8 d-lysine residues. All constructs were labeled with Rhodamine B to enable quantitative cellular uptake analysis. Flow cytometry and confocal microscopy in OVCAR-8 ovarian cancer cells revealed that the PNA incorporating six positively charged purines (MCP6, multiple charged purines with an overall 6 positive charges) exhibited markedly enhanced cellular internalization compared to both the other MCP-PNAs and the CPP-PNA controls. MCP-PNAs showed no noticeable signs of cell toxicity, and their binding affinities (thermal melting profiles) were comparable to CPP-PNAs. In addition, MCP-PNAs well discriminated single mismatches in RNA, similarly to CPP-PNAs. Overall, this strategy provides a simple and effective approach for generating inherently cell-permeable PNAs.