Soudah Terese, Khawaled Saleh, Aqeilan Rami I, Yavin Eylon
The Institute for Drug Research, The School of Pharmacy, and Lautenberg Center for Immunology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University of Jerusalem, Hadassah Ein-Kerem, Jerusalem 9112102, Israel.
ACS Omega. 2019 Aug 12;4(9):13954-13961. doi: 10.1021/acsomega.9b01697. eCollection 2019 Aug 27.
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-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-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-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.
将核酸高效递送至细胞仍然是一项巨大的挑战。肽核酸（PNA）是具有中性主链的DNA类似物，通过固相肽化学合成。这使得可以通过直接的合成路线将线性短肽（即细胞穿透肽）引入PNA分子，作为促进PNA细胞摄取的一种手段。在此，我们设计了一种合成路线，其中在固相载体上制备环肽，并用PNA分子进行延伸，所有合成均在固相上完成。这使得环肽与PNA分子的缀合在环肽-PNA缀合物（C-PNA）从固相载体上切割后仅需一步纯化。所选的PNA序列是一种与成熟miR-155互补的抗miR-155分子，miR-155是一种公认的致癌性miRNA。通过用异硫氰酸荧光素标记C-PNA，我们观察到在低浓度（0.5μM）下其能高效摄取到胶质母细胞瘤细胞（U87MG）中，荧光激活细胞分选（FACS）分析和共聚焦显微镜证实了这一点。FACS分析还表明摄取机制是能量依赖的。最后，通过定量逆转录聚合酶链反应分析miR155水平，并观察U87MG细胞中细胞活力和增殖的降低（XTT和集落形成试验证实），显示了C-PNA的抗miR活性。鉴于环肽与线性肽相比具有更高的生物学稳定性，这种合成方法可能是合成环肽-PNA缀合物的一种有用且直接的方法。
