Robust visualization of membrane protein by aptamer mediated proximity ligation assay and Förster resonance energy transfer.

Abstract

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.