Wilbanks Brandon, Beimers William, Dugan Maria, Weiskittel Taylor, Maher L J
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.
Present address: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
PNAS Nexus. 2023 May 4;2(5):pgad151. doi: 10.1093/pnasnexus/pgad151. eCollection 2023 May.
The efficient and specific delivery of functional cargos such as small-molecule drugs, proteins, or nucleic acids across lipid membranes and into subcellular compartments is a significant unmet need in nanomedicine and molecular biology. Systematic Evolution of Ligands by EXponential enrichment (SELEX) exploits vast combinatorial nucleic acid libraries to identify short, nonimmunogenic single-stranded DNA molecules (aptamers) capable of recognizing specific targets based on their 3D structures and molecular interactions. While SELEX has previously been applied to identify aptamers that bind specific cell types or gain cellular uptake, selection of aptamers capable of carrying cargos to specific subcellular compartments is challenging. Here, we describe peroxidase proximity selection (PPS), a generalizable subcellular SELEX approach. We implement local expression of engineered ascorbate peroxidase APEX2 to biotinylate naked DNA aptamers capable of gaining access to the cytoplasm of living cells without assistance. We discovered DNA aptamers that are preferentially taken up into endosomes by macropinocytosis, with a fraction apparently accessing APEX2 in the cytoplasm. One of these selected aptamers is capable of endosomal delivery of an IgG antibody.
将小分子药物、蛋白质或核酸等功能性货物有效且特异性地递送至脂质膜并进入亚细胞区室，是纳米医学和分子生物学中一个尚未得到满足的重大需求。指数富集配体系统进化技术（SELEX）利用大量组合核酸文库来鉴定短的、非免疫原性的单链DNA分子（适体），这些适体能够基于其三维结构和分子相互作用识别特定靶标。虽然SELEX此前已被用于鉴定结合特定细胞类型或实现细胞摄取的适体，但选择能够将货物携带至特定亚细胞区室的适体具有挑战性。在此，我们描述了过氧化物酶邻近选择（PPS），一种可推广的亚细胞SELEX方法。我们实现了工程化抗坏血酸过氧化物酶APEX2的局部表达，以生物素化能够在无辅助情况下进入活细胞胞质的裸露DNA适体。我们发现了通过巨胞饮作用优先被摄取到内体中的DNA适体，其中一部分显然进入了胞质中的APEX2。这些所选适体之一能够将IgG抗体递送至内体。
