Protein Delivery Using Cys₂His₂ Zinc-Finger Domains

The development of new methods for delivering proteins into cells is a central challenge for advancing both basic research and therapeutic applications. We previously reported that zinc-finger nuclease proteins are intrinsically cell-permeable due to the cell-penetrating activity of the Cys2−His2 zinc-finger domain. Here, we demonstrate that genetically fused zincfinger motifs can transport proteins and enzymes into a wide range of primary and transformed mammalian cell types. We show that zinc-finger domains mediate protein uptake at efficiencies that exceed conventional protein transduction systems and do so without compromising enzyme activity. In addition, we demonstrate that zinc-finger proteins enter cells primarily through macropinocytosis and facilitate high levels of cytosolic delivery. These findings establish zincfinger proteins as not only useful tools for targeted genome engineering but also effective reagents for protein delivery. T efficient delivery of proteins and enzymes into cells is an integral step for disease treatment and many basic research applications. Among the safest approaches for delivering these macromolecules into cells is direct delivery of purified protein. To date, numerous approaches have been developed that facilitate direct protein entry into the cytoplasm of mammalian cells, including isolated or designed cellpenetrating peptides, liposomes, protein containers, polymeric microspheres, and nanoparticles, naturally occurring and engineered “supercharged” proteins, and viruslike particles. However, these approaches are routinely confounded by various factors, such as low uptake efficiency, unfavorable endosomal escape properties, poor stability, inadvertent cell-type dependency, cytotoxicity, and compromised enzyme activity. As such, the development of new methods that enable the safe and efficient delivery of purified proteins into cells is needed to fulfill the promise of protein delivery as a therapeutic modality. We previously showed that zinc-finger nucleases (ZFNs) chimeric enzymes that induce DNA double-strand breaks at targeted genomic loci and thus promote genome editing27are intrinsically cell-permeable. The source of this cell-penetrating activity was shown to be the Cys−His2 zinc-finger domain. Cys2−His2 zinc-fingers are among the most common DNAbinding motifs across all domains of life and consist of approximately 30 amino acid residues situated within a ββα structure (Figure 1A). Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, zinc-finger proteins have emerged as powerful tools for genome engineering. In addition, because zincfingers are inherently cell-permeablepresumably due to their net positive chargewe hypothesized that they could serve as generalized protein transduction reagents and facilitate direct protein delivery into various mammalian cell types, as observed with ZFN proteins. Here, we show that genetic fusion of zincfingers (hereafter referred to as ZiF domains) onto proteins imparts a high degree of cell-penetrating activity that exceeds conventional protein transduction systems and that ZiF domains mediate efficient uptake of proteins and enzymes into a variety of primary and transformed mammalian cell types. In order to determine whether ZiF domains could deliver functional proteins into mammalian cells, we genetically fused one-, two-, three-, four-, fiveor six-finger ZiF proteins to the N-terminus of Emerald GFP (EmGFP), an engineered GFP variant that displays improved photostability and brightness compared to enhanced GFP. Unlike “supercharged” GFP, which possesses the innate ability to cross cell membranes due to its high overall charge, EmGFP has no cell-penetrating activity. Because each ZiF domain harbors positive charge (theoretical net charge for typical oneand six-finger ZiF domains at physiological pH: +4 and +20, respectively), we anticipated that ZiF proteins composed of extended zinc-finger arrays would demonstrate increased cell-penetrating activity in comparison to those with fewer. To ensure that ZiF proteins could not recognize DNA, we substituted the α-helical DNAbinding residues of each zinc-finger domain (i.e., −1, 2, 3, and 6) with alanine (Figure 1A). Importantly, because these substitutions lie within positions that routinely encode positively charged residues, the overall net charge for each ZiF domain was expected to decrease (theoretical net charge for an alanine-substituted six-finger ZiF protein: +10. We note that theoretical net charges were calculated in the absence of terminal modifications, such as the polyhistidine-tag, which are expected to increase charge). The complete amino acid Received: April 16, 2014 Accepted: June 17, 2014 Published: June 17, 2014 Letters pubs.acs.org/acschemicalbiology © 2014 American Chemical Society 1662 dx.doi.org/10.1021/cb500282g | ACS Chem. Biol. 2014, 9, 1662−1667 Terms of Use