AntimiR-155 Cyclic Peptide-PNA Conjugate: Synthesis, Cellular Uptake, and Biological Activity

Abstract

Efficient delivery of nucleic acids into cells still remains a great challenge. Peptide nucleic acids (PNAs) are DNA analogues with a neutral backbone and are synthesized by solid phase peptide chemistry. This allows a straightforward synthetic route to introduce a linear short peptide (a.k.a. cell-penetrating peptide) to the PNA molecule as a means of facilitating cellular uptake of PNAs. Herein, we have devised a synthetic route in which a cyclic peptide is prepared on a solid support and is extended with the PNA molecule, where all syntheses are accomplished on the solid phase. This allows the conjugation of the cyclic peptide to the PNA molecule with the need of only one purification step after the cyclic peptide-PNA conjugate (C₉-PNA) is cleaved from the solid support. The PNA sequence chosen is an antimiR-155 molecule that is complementary to mature miR-155, a well-established oncogenic miRNA. By labeling C₉-PNA with fluorescein isothiocyanate, we observe efficient cellular uptake into glioblastoma cells (U87MG) at a low concentration (0.5 μM), as corroborated by fluorescence-activated cell sorting (FACS) analysis and confocal microscopy. FACS analysis also suggests an uptake mechanism that is energy-dependent. Finally, the antimiR activity of C₉-PNA was shown by analyzing miR155 levels by quantitative reverse transcription polymerase chain reaction and by observing a reduction in cell viability and proliferation in U87MG cells, as corroborated by XTT and colony formation assays. Given the added biological stability of cyclic versus linear peptides, this synthetic approach may be a useful and straightforward approach to synthesize cyclic peptide-PNA conjugates.

Scheme 1. Solid-Phase Synthesis of C₉–PNAs

Figure 1

miR-155 expression following incubation of PNA conjugates (0.5 μM) in U87MG cells for 24 h at 37 °C. miR-155 is shown in comparison to scrambled PNA controls. **P value < 0.01.

Figure 2

Cell viability for U87GM cells and THSCs cells as determined by the XTT assay. (a) U87GM cells were treated with 1 μM PNA conjugates for 72 h at 37 °C (in triplicates in 96-well plates). Viability is shown in comparison to scrambled PNA controls. *P value < 0.05, ***P value < 0.001. (b) THSCs cells were treated with 1 μM PNA conjugates for 72 h at 37 °C (in triplicates in 96-well plates). Viability is shown in comparison to scrambled PNA controls. *P value < 0.05.

Figure 3

C₉–PNA reduces the colony survival of U87MG glioblastoma cells. Colony formation assay of cells treated with 1.0 μM of either dK₄–PNA and C₉–PNA or scrambled controls (dK₄–PNA-Scr and C₉–PNA-Scr). After 2 weeks, the plates were fixed and stained, and the colonies were counted (in triplicates). *P value < 0.05.

Figure 4

C₉–PNA–FITC shows efficient cellular uptake into U87MG cells. (A) Confocal images of C₉–PNA–FITC and dK₄–PNA–FITC (at 0.5 μM) after a short incubation of 3 h at 37 °C. (B) FACS analysis of C₉–PNA–FITC and dK₄–PNA–FITC (at 0.5 μM) cellular uptake into U87MG cells after 2 h of incubation at 37 °C.

Figure 5

C₉–PNA–FITC cellular uptake is energy-dependent. C₉-PNA–FITC uptake into U87MG cells after a 2 h incubation (A) at 4 °C or (B) at 37 °C with 10 mM NaN₃/10 mM 2-deoxy-d-glucose.

Figure 6

C₉–PNA–FITC cellular uptake is predominantly in the cytoplasm. Confocal microscopy images of U87MG glioblastoma cells incubated with C₉–PNA–FITC or dK₄–PNA–FITC (at 0.5 μM) after an incubation of 3 h at 37 °C. (A) PNA–FITC alone (green); (B) overlay of PNA–FITC (green) with Lyso-Tracker Red (red) staining of lysosome (C); overlay of PNA–FITC (green) with Hoechst (blue) staining of nuclei; and (D) overlay of both stains with PNA–FITC.