ja9b13621_si_001.pdf (21.6 MB)
Cell-Penetrating Streptavidin: A General Tool for Bifunctional Delivery with Spatiotemporal Control, Mediated by Transport Systems Such as Adaptive Benzopolysulfane Networks
journal contribution
posted on 2020-02-28, 17:03 authored by Javier López-Andarias, Jacques Saarbach, Dimitri Moreau, Yangyang Cheng, Emmanuel Derivery, Quentin Laurent, Marcos González-Gaitán, Nicolas Winssinger, Naomi Sakai, Stefan MatileIn 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.