Cell-Penetrating Streptavidin: A General Tool for Bifunctional Delivery with Spatiotemporal Control, Mediated by Transport Systems Such as Adaptive Benzopolysulfane Networks

Abstract

In this report, cell-penetrating streptavidin (CPS) is introduced to exploit the full power of streptavidin−biotin biotechnology in cellular uptake. For this purpose, transporters, here cyclic oligochalcogenides (COCs), are covalently attached to lysines of wild-type streptavidin. This leaves all four biotin binding sites free for at least bifunctional delivery. To maximize the standards of the quantitative evaluation of cytosolic delivery, the recent chloroalkane penetration assay (CAPA) is coupled with automated high content (HC) imaging, a technique that combines the advantages of fluorescence microscopy and flow cytometry. According to the resulting HC-CAPA, cytosolic delivery of CPS equipped with four benzopolysulfanes was the best among all tested CPSs, also better than the much smaller TAT peptide, the original cell-penetrating peptide from HIV. HaloTag-GFP fusion proteins expressed on mitochondria were successfully targeted using CPS carrying two different biotinylated ligands, HaloTag substrates or anti-GFP nanobodies, interfaced with peptide nucleic acids, flipper force probes, or fluorescent substrates. The delivered substrates could be released from CPS into the cytosol through desthiobiotin−biotin exchange. These results validate CPS as a general tool which enables unrestricted use of streptavidin−biotin biotechnology in cellular uptake.

Automated high-content imaging for cellular uptake, from the Schmuck cation to the latest cyclic oligochalcogenides

“…The CP 50 value of 7.3 ± 0.5 µM obtained for 25 outperformed the CP 50 value of 14.9 ± 0.5 μM of the previously reported cell-penetrating streptavidin, in which four asparagusic acids are covalently bound to the protein through irreversible triazole linkages [ 59 ]. The CP 50 value of 7.3 ± 0.5 µM of 25 was not far above the CP 50 value of 3.1 ± 0.5 µM of HIV Tat, the original CPP [ 55 ].…”

“…In a CAPA, the final fluorescence response is usually recorded by flow cytometry [55,62]. In a HC CAPA, flow cytometry is replaced by HC automated microscopy, using multiwell plates and registering data on the fluorescence intensity and fluorescence localization in thousands of cells per condition, at HT [56][57][58][59]. To properly assess its potential, automated HC imaging was optimized first.…”

“…The difference between the optimized HGM cells and the unoptimized transfected cells is again best appreciated with the naked eye: Whereas the improvement of the automated selection from Figure 8a to Figure 8b is not visible on the first view, the improvement from Figure 11a to Figure 11d is massive, and Figure 11d starts to resemble more the quasiideal Figure 8b. The automated removal of 80% of all cells to obtain Figure 11d (compared to 15% to produce Figure 8b) is not problematic because HC microscopy registers, screens, and evaluates the data after the optimization of the ideal conditions for the analysis [56][57][58][59]. Figure 10a), b) all cells inside the punctate regions with a high GFP signal, considered as the Golgi apparatus (yellow areas in Figure 10a), c) selected cells inside the cell body mask (light and dark blue areas in Figure 10c), and d) punctate regions with a high GFP signal in selected cells, considered as the Golgi apparatus (yellow areas in Figure 10c).…”

“…(b) The CP 50 value of 25 with standard errors using the cell body mask (blue) and the mitochondrial mask (yellow). The statistical significance was determined using the one-tailed paired Student's t-test: *p < 0.05.The CP 50 value of 7.3 ± 0.5 µM obtained for 25 outperformed the CP 50 value of 14.9 ± 0.5 μM of the previously reported cellpenetrating streptavidin, in which four asparagusic acids are covalently bound to the protein through irreversible triazole linkages[59]. The CP 50 value of 7.3 ± 0.5 µM of 25 was not far above the CP 50 value of 3.1 ± 0.5 µM of HIV Tat, the original CPP[55]…”

“…Recently, the CAPA has been introduced to quantify cytosolic delivery [ 55 ]. The combination with image-based high-content (HC) screening [ 56 – 58 ] has been suggested to further improve the standards set by the CAPA [ 59 ]. Standard high-throughput (HT) screening has been used regularly to facilitate studies on cellular uptake [ 60 ].…”