Patterning protein complexes on DNA nanostructures using a GFP nanobody

Abstract

DNA nanostructures have become an important and powerful tool for studying protein function over the last 5 years. One of the challenges, though, has been the development of universal methods for patterning protein complexes on DNA nanostructures. Herein, we present a new approach for labeling DNA nanostructures by functionalizing them with a GFP nanobody. We demonstrate the ability to precisely control protein attachment via our nanobody linker using two enzymatic model systems, namely adenylyl cyclase activity and myosin motility. Finally, we test the power of this attachment method by patterning unpurified, endogenously expressed Arp2/3 protein complex from cell lysate. By bridging DNA nanostructures with a fluorescent protein ubiquitous throughout cell and developmental biology and protein biochemistry, this approach significantly streamlines the application of DNA nanostructures as a programmable scaffold in biological studies.

SNAP-Tagged Nanobodies Enable Reversible Optical Control of a G Protein-Coupled Receptor via a Remotely Tethered Photoswitchable Ligand

“…While the ability to target NBs directly to wild-type, endogenous proteins of interest offers great potential, the targeting of proteins with incorporated GFP tags also offers many advantages. Strong antibodies or nanobodies with sufficient strength and specificity are not available for many antigens but GFP, for which high affinity nanobodies have been developed, extensively characterized and applied in vitro and in living cells 11,29,30 has been successfully incorporated into many proteins, often in multiple different positions, that have been well-characterized functionally 31. In addition, recent genetic advances based on the CRISPR-Cas9 system have made it increasingly feasible to incorporate GFP or other tags into native proteins either at the transgenic level or even in postmitotic cells in the nervous system.…”

SNAP-tagged nanobodies enable reversible optical control of a G protein-coupled receptor via a remotely tethered photoswitchable ligand

“…While the ability to target NBs directly to wild-type, endogenous proteins of interest offers great potential, the targeting of proteins with incorporated GFP tags also offers many advantages. Strong antibodies or nanobodies with sufficient strength and specificity are not available for many antigens but GFP, for which exceptionally high affinity nanobodies have been developed, extensively characterized and applied in vitro and in living cells [13, 27] [28] has been successfully incorporated into many proteins, often in multiple different positions, that have been well-characterized functionally[29]. In addition, recent genetic advances based on the CRISPR-Cas9 system have made it increasingly feasible to incorporate GFP or other tags into native proteins either at the transgenic level or even in post-mitotic cells in the nervous system[30] [31].…”

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC

“…An N-terminal mCitrine version of adaptor MIRs with a FLAG tag was used for bimolecular FRET assays. A GFP nanobody construct with C-terminal SNAP and FLAG tags in pBiex1 vector were used to bind GFP-tagged proteins as reported by us previously (17,37,38).…”