DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K⁺ Channels

Abstract

In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K channel proteins are assembled through zinc-finger protein (ZFP)-adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP-fused Kir3 channels and ZFP-based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells.

Emerging applications at the interface of DNA nanotechnology and cellular membranes: Perspectives from biology, engineering, and physics

“…When a heterotetramer binding site configuration is used, a roughly threefold increase in whole-cell K + currents is observed when compared to DNA constructs with no binding sites. 175 Reproduced with permission from Kurakawa et al , Angew. Chem., Int.…”

DNA Aptamers for the Functionalisation of DNA Origami Nanostructures

“…Enzymatic activity resulting in the generation of xylulose and NADH could also be controlled through modulating inter-enzyme distances. Recent work from the same group also demonstrated the use of zinc-finger protein adaptors consisting of single-stranded hairpin structures for the binding of Kir3 K + channel proteins [69]. The precise fabrication allowed the design of various cavities in the DNA origami such that the K + channel current activity could be controlled by the oligomerisation state of the protein complex.…”

DNA Aptamers for Functionalisation of DNA Origami Nanostructures

“…The Morii group has developed Zinc-finger [59] or leucine-zipper [60] controlled through modulating inter-enzyme distances. Recent work from the same group also demonstrated the use of zinc-finger protein adaptors consisting of single-stranded hairpin structures for the binding of Kir3 K + channel proteins [62]. The precise fabrication allowed design of various cavities in the DNA origami such that K + channel current activity could be controlled by the oligomerization state of the protein complex.…”