Department of Chemistry, Binghamton University, The State University of New York, Binghamton, New York, USA.
Department of Biological Sciences, Binghamton University, The State University of New York, Binghamton, New York, USA.
Biopolymers. 2022 Apr;113(4):e23484. doi: 10.1002/bip.23484. Epub 2021 Dec 16.
Cell-penetrating peptides (CPPs) have been extensively used to deliver peptide nucleic acid (PNA) in cells. We have previously found that replacement of cytosine in triplex-forming PNAs with 2-aminopyridine (M) not only enhanced RNA binding, but also improved cellular uptake of PNAs. In this study, we used confocal fluorescence microscopy to evaluate the ability of CPPs to further improve cellular uptake of M-modified PNAs. We found that PNAs conjugated with Tat and octa-arginine peptides were effectively taken up in MCF7 cells when supplied in cell media at 1 μM. Remarkably, M-modified PNA without any CPP conjugation also showed strong uptake when the concentration was increased to 5 μM. Majority of PNA conjugates remained localized in distinct cytoplasmic vesicles, as judged by dot-like fluorescence patterns. However, M-modified PNAs conjugated with Tat, octa-arginine, and even a simple tri-lysine peptide also showed dispersed fluorescence in cytoplasm and were taken up in nuclei where they localized in larger vesicles, most likely nucleoli. Endosomolytic peptides or chemicals (chloroquine and CaCl ) did not release the conjugates from cytosolic vesicles, which suggested that the PNAs were not entrapped in endosomes. We hypothesize that M-modified PNAs escape endosomes and accumulate in cellular compartments rich in RNA, such as nucleoli, stress granules, and P-bodies.
细胞穿透肽 (CPPs) 已被广泛用于将肽核酸 (PNA) 递送到细胞中。我们之前发现，用 2-氨基吡啶 (M) 替代三链形成 PNAs 中的胞嘧啶不仅增强了 RNA 结合，还提高了 PNAs 的细胞摄取。在这项研究中，我们使用共聚焦荧光显微镜来评估 CPP 进一步提高 M 修饰 PNAs 细胞摄取的能力。我们发现，当在细胞培养基中以 1 μM 的浓度提供时，与 Tat 和八聚精氨酸肽缀合的 PNAs 可有效地被 MCF7 细胞摄取。值得注意的是，当浓度增加到 5 μM 时，没有任何 CPP 缀合的 M 修饰 PNA 也表现出强烈的摄取。大多数 PNA 缀合物仍定位于明显的细胞质小泡中，这可以通过点状荧光模式来判断。然而，与 Tat、八聚精氨酸缀合的 M 修饰 PNAs 甚至与简单的三赖氨酸肽缀合的 PNA 也在细胞质中显示弥散荧光，并被摄取到核内，在核内它们定位于较大的小泡中，很可能是核仁。溶酶体解聚肽或化学物质（氯喹和 CaCl）不能从小泡中释放出缀合物，这表明 PNA 未被内体困住。我们假设 M 修饰的 PNAs 逃避内体并在富含 RNA 的细胞区室中积累，例如核仁、应激颗粒和 P 体。
