His-Tag-Aptamer-Liposome-Conjugates as a Multipurpose Tool for Recombinant Protein Screening and Protein-Based Bioassays

Liposome-aptamer conjugates are powerful tools for bioanalysis and drug delivery by taking advantage of aptamer binding specificity and liposome encapsulation capabilities. Here, we developed multipurpose conjugates that specifically and effectively bind to his-tags and demonstrated their capability as screening tool for his-tagged proteins and as signaling tool for bioassays. Using the poly histidine-tag (his-tag) binding aptamer NDM1-H14-01 (his-Apt), we studied aptamer-liposome immobilization with special focus on retaining aptamer functionality and liposome integrity. This involved various strategies for modifying liposomes with aptamers, i.e., cholesterol-functionalized aptamers were inserted into the liposome pre- or post-synthesis, amine-modified aptamers were covalently coupled to COOH-groups on the liposome surface, and biotinylated aptamers were incubated with streptavidin-modified liposomes, respectively. Extensive characterization of liposome and aptamer properties, as well as conjugate functionality via heterogeneous binding assays, proved post-insertion and biotinylated aptamers successful with a stability of at least 4 months at 4 °C, whereas the other methods destabilized the aptamer structure. The successful conjugates were further optimized to (i) serve as a tool for easy detection and screening of his-tag accessibility in recombinant proteins, which can replace common metal-based formats (ii) enable efficient site-directed immobilization of proteins onto liposomes while omitting the need of additional protein modifications, and (iii) demonstrate that aptamers can function as a reliable secondary recognition element for protein-modified liposomes, e.g., as internal control system. Consequently, these highly functional and stable his-Apt-liposome conjugates represent a versatile platform technology for bioassays, which can substitute labor- and cost-intensive measurements, as well as improve assay control and reproducibility.