A DNA nanoassembly-based approach to map membrane protein nanoenvironments

Abstract

Super-resolution imaging has revealed that most proteins at the plasma membrane are not uniformly distributed but localize to dynamic domains of nanoscale dimensions. To investigate their functional relevance, there is a need for methods that enable comprehensive mapping of the compositions and spatial organizations of membrane protein nanodomains in cell populations. However, current superresolution methods are limited to analysing small, preselected subsets of proteins, at very low sampling fractions. Here we describe the development of a non-microscopy based super-resolution method for unbiased ensemble analysis of membrane protein nanodomains. The method, termed NANOscale DEciphEring of membrane Protein nanodomains (NanoDeep), is based on the use of DNA nanoassemblies to translate membrane protein organization information into a DNA sequencing readout. Using NanoDeep, we characterized the nanoenvironments of Her2, a membrane receptor of critical relevance in cancer. We found that the occupancies of Her2, Her3 and EGFR in the nanoenvironments surrounding Her2 were similar in two cell lines with vastly different expression levels of Her2. Further, we found that adding Heregulin-β1 to cancer cells led to increased occupancy of Her2 and Her3, and to a lesser extent EGFR, in Her2 nanoenvironments. NanoDeep has the potential to provide new insights into the roles of the composition and spatial organization of protein nanoenvironments in the regulation of membrane protein function. MainCells sense extracellular signals, such as protein ligands, through specialized proteins present on the cell surface called membrane receptors. The protein nanoenvironment, i.e. the composition and spatial organization of proteins surrounding membrane receptors, is dynamic and often modulated by ligand binding, suggesting that it has functional relevance 1, 2 . Super-resolution microscopy has enabled the characterization of the nanoscale spatial distributions of proteins at the cell membrane but is limited to simultaneously analysing only a few proteins. Further, using super-resolution microscopy, it is only feasible to image a small fraction of all protein nanodomains present in a cell population 3-5 . DNA detection as a proxy for protein detection through the use of oligo-conjugated affinity binders has been used extensively for signal amplification 6 and analysis of proximity between pairs of proteins 7, 8 . Further, DNA sequencing is used as a readout in DNA microscopy, a new method to visualize the spatial organization of RNA and DNA molecules inside cells 9-11 . Here we present NanoDeep, a method that uses DNA sequencing to decipher the nanoscale spatial distribution of membrane proteins. This method allows for the detection en masse of the inventory of proteins that forms the nanoenvironment of any reference membrane protein in cell populations. We demonstrate the application of NanoDeep to the analysis of protein nanoenviroments surrounding Human Epidermal Growth Factor Receptor 2 (Her2). Her2 cooperates with members of the Ep...

Obtaining Precise Molecular Information via DNA Nanotechnology

“…Although there are many well-constructed methods for studying PPIs in vitro, it is still difficult to obtain PPIs in situ, such as on cell membranes or inside the cells. Ambrosetti et al developed a non-microscopy-based method for ensemble analysis of membrane proteins’ interactions in situ [ 10 ] ( Figure 4 C). Their method was based on the use of a DNA nanostructure (termed NanoComb) to locally encode membrane protein spatial distribution information into a newly synthesized sequence that could be read later by DNA sequencing.…”

“…( B ) Structure of the junctured-DNA tweezer (left) and the strategy used to attach a given protein at a given tip (right), adapted with permission from [ 107 ]. ( C ) Schematic of the DNA NanoComb method, adapted with permission from [ 10 ].…”

“…Utilizing these single-molecule techniques, researchers are able to reconstruct the structures of biomolecules such as proteins at an atomic-level resolution [ 2 , 5 ], and accurately investigate intermolecular interactions such as protein–protein interactions (PPIs) [ 6 , 7 ] and protein–DNA interactions [ 8 ]. These advancements are rapidly transforming our understanding of the structure of biomolecules [ 9 ], as well as biological processes [ 10 ].…”

Modular DNA-origami-based nanoarrays enhanced cell binding-affinity through “lock-key” interaction

“…Notably, the dynamic distribution of protein receptors is usually nonuniform and discontinuous, with the low expression of some proteins, which make it difficult to accurately identify and isolate targets [8][9][10][11] . Additionally, some membrane protein receptors prefer to cluster to specific membrane domains [12][13][14][15] , and even form high-order clusters or coexist with adjacent proteins to regulate functions, such as EpCAM 16,17 , EGFR 18 and HER2 19 . Traditional strategies generally use magnetic beads modified antibodies or aptamers to achieve cell recognition and binding, but their disordered arrangement may lead to steric hindrance, low binding-affinity, and lack of cellular information expressing heterogeneous proteins [20][21][22] .…”

Nanoscale Organization of FasL on DNA Origami as a Versatile Platform to Tune Apoptosis Signaling in Cells

“…DNA origami, on the other hand, is a powerful tool to build versatile DNA-based platforms [12][13][14][15] which satisfy multiple structural and biofunctional constraints including the possibility of complex molecular conjugation with biomolecules such as peptides or proteins with nanometric precision. [16][17][18][19][20][21][22][23] Such defined molecular organization can provide unique insights into ligand-receptor interactions in signaling complexes and the resulting signal transduction as well as serve to enhance or block particular signals. [24][25] Among the signaling complexes which present themselves as supramolecular assemblies, receptors and ligands of the tumor necrosis factor (TNF) super family are extensively studied and models of their multimerization and cluster formation have been proposed.…”