Li Ying, Qian Meiqi, Cheng Yuping, Qiu Xue
Key Laboratory of Marine Drug, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China.
Key Laboratory of Marine Drug, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China; Laboratory for Marine Drugs and Bioproducts, Qingdao Marine Science and Technology Center, Qingdao 266237, China.
Colloids Surf B Biointerfaces. 2025 Apr;248:114486. doi: 10.1016/j.colsurfb.2024.114486. Epub 2024 Dec 30.
In situ cell imaging plays a crucial role in studying physiological and pathological processes of cells. Proximity ligation assay (PLA) and rolling circle amplification (RCA) are commonly used to study the abundance and interactions of biological macromolecules. The most frequently applied strategy to visualize the RCA products is with single-fluorophore probe, however, cellular auto-fluorescence and unbound fluorescent probes could interfere with RCA products, leading to non-specific signals. Here, we present a novel approach combining aptamer mediated PLA, RCA, and Förster Resonance Energy Transfer (FRET), namely Apt-PLA-RCA-FRET, for sensitive in situ imaging and analysis of the abundances and interactions of membrane proteins such as tetraspanin CD63 and human epidermal growth factor receptor 2 (HER2). Apt-RCA-FRET was initially designed to show its ability to assess the abundance of target proteins on different cells. Dual functional oligonucleotides served as both the aptamer for recognizing specific membrane proteins and the primer of circular DNA for following RCA process, and the resulting RCA products were subsequently imaged by FRET signals from Cy3 to Cy5 probes which hybridized sequentially on them. FRET was demonstrated to show its great potential to resist the interferences of nonspecific fluorescence compared to single-fluorophore strategies. PLA was then introduced to Apt-RCA-FRET to investigate the spatial localization of different proteins on cell membrane and their interactions. Our approach utilizing aptamer as membrane proteins recognition element simply converted the abundance of proteins into nucleic acid signals and facilitated the following signal amplification, thus it serves as an important alternative to methods typically based on antibody and presents a more robust and sensitive method for analyzing the abundances of different cell membrane proteins and their spatial localization, which offers valuable insights into physiological and pathological processes of cells.
原位细胞成像在研究细胞的生理和病理过程中起着至关重要的作用。邻近连接分析（PLA）和滚环扩增（RCA）常用于研究生物大分子的丰度和相互作用。可视化RCA产物最常用的策略是使用单荧光团探针，然而，细胞自发荧光和未结合的荧光探针可能会干扰RCA产物，导致非特异性信号。在此，我们提出了一种将适体介导的PLA、RCA和荧光共振能量转移（FRET）相结合的新方法，即Apt-PLA-RCA-FRET，用于对四跨膜蛋白CD63和人表皮生长因子受体2（HER2）等膜蛋白的丰度和相互作用进行灵敏的原位成像和分析。Apt-RCA-FRET最初被设计用于展示其评估不同细胞上靶蛋白丰度的能力。双功能寡核苷酸既作为识别特定膜蛋白的适体，又作为后续RCA过程中环状DNA的引物，随后通过依次杂交在其上的Cy3至Cy5探针的FRET信号对产生的RCA产物进行成像。与单荧光团策略相比，FRET被证明具有抵抗非特异性荧光干扰的巨大潜力。然后将PLA引入Apt-RCA-FRET，以研究不同蛋白质在细胞膜上的空间定位及其相互作用。我们利用适体作为膜蛋白识别元件的方法简单地将蛋白质的丰度转化为核酸信号，并促进了后续的信号放大，因此它是通常基于抗体的方法的重要替代方法，为分析不同细胞膜蛋白的丰度及其空间定位提供了一种更强大、更灵敏的方法，这为细胞的生理和病理过程提供了有价值的见解。
