Cholesterol- and ssDNA-binding fusion protein-mediated DNA tethering on the plasma membrane

DNA modification of the plasma membrane is an excellent approach for controlling membrane-protein interactions, modulating cell-cell/cell-biomolecule interactions, and extending the biosensing field. The hydrophobic insertion of DNA conjugated with hydrophobic anchoring molecules is utilized for tethering DNA on the cell membrane. In this study, we developed an alternative approach to tether DNA on the plasma membrane based on ssDNA- and cholesterol-binding proteins. We designed a fusion protein (Rep-ALOD4) composed of domain 4 of anthrolysin O (ALOD4), which binds to cholesterol in the plasma membrane, and a replication initiator protein derived from porcine circovirus type 2 (Rep), which forms covalent bonds with single-stranded DNA (ssDNA) with a Rep recognition sequence. Rep-ALOD4 conjugates ssDNA to Rep and binds to the plasma membrane <i>via</i> cholesterol, thus tethering ssDNA to the cells. Quartz crystal microbalance measurements showed that membrane cholesterol binding of Rep-ALOD4 to the lipid bilayer containing cholesterol was accelerated above 20% (w/w) cholesterol in the lipid bilayer. Rep-ALOD4 was conjugated to fluorescein-labeled ssDNA (S-FITC-Rep-ALOD4) and used to treat human cervical tumor HeLa cells. The green signal assigned to S-FITC-Rep-ALOD4 was detected along HeLa cells, whereas diminished by cholesterol removal with methyl β-cyclodextrins. Moreover, ssDNA-conjugated Rep-ALOD4 tethered ssDNA-conjugated functional proteins on the HeLa cell plasma membrane <i>via</i> complementary base pairing. Collectively, Rep-ALOD4 has the potential as an ssDNA-tethering material <i>via</i> plasma membrane cholesterol to extend cell surface engineering.