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 (C 9 –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 C 9 –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 C 9 –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.

Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications

“…Searching for a general membrane transporter for therapeutic agents, Pei and co-workers discovered that cyclic peptides were ≈20-fold more efficient for cytosolic delivery in HeLa cells compared to common CPPs, such as, Tat and R 9 [202]. Yavin and co-workers adopted this strategy and synthesized a PNA conjugate with a cyclic peptide C 9 -PNA (Figure 15) [203]. After incubation at 500 nM for 3 h, C 9 -PNA showed significant uptake in U87MG cells as judged by live cell fluorescence microscopy and FACS analysis, compared to less efficient uptake of K 4 -PNA under the same conditions.…”

Fluorescent cyclic peptides for cell imaging

“…Yavin and coworkers synthesized FITC‐labeled peptide nucleic acids with a cyclic structure and confirmed their uptake in U87MG glioblastoma cells as well as their blocking function of miR‐155, a reported oncogenic miRNA. [ 83 ]…”

“…Yavin and coworkers synthesized FITC-labeled peptide nucleic acids with a cyclic structure and confirmed their uptake in U87MG glioblastoma cells as well as their blocking function of miR-155, a reported oncogenic miRNA. [83] Cyclic fluorescent peptides have also been developed to target liposomes. In the work reported by Dreher and coworkers, a novel cyclic peptide including the NGR motif (cKNGRE) was conjugated to the pH-sensitive Oregon Green dye.…”

Exploring miR-9 Involvement in Ciona intestinalis Neural Development Using Peptide Nucleic Acids

“…A set of unique features, including high specific and selective binding affinity towards complementary DNA and RNA strands [11] and enzymatic resistance [12], makes PNAs particularly suitable for antisense therapy [13]. At present, data on the use of PNAs to target miRNAs are accumulating [14][15][16][17][18][19][20], demonstrating the potential of PNA for future therapeutic applications, as well as for studying miRNA functions. However, only a few studies have been performed in vivo.…”