Nanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in Cells

Abstract

Cell signaling is initiated by characteristic protein patterns in the plasma membrane, but tools to decipher their molecular organization and activation are hitherto lacking. Among the well‐known signaling pattern is the death inducing signaling complex with a predicted hexagonal receptor architecture. To probe this architecture, DNA origami‐based nanoagents with nanometer precise arrangements of the death receptor ligand FasL are introduced and presented to cells. Mimicking different receptor geometries, these nanoagents act as signaling platforms inducing fastest time‐to‐death kinetics for hexagonal FasL arrangements with 10 nm inter‐molecular spacing. Compared to naturally occurring soluble FasL, this trigger is faster and 100× more efficient. Nanoagents with different spacing, lower FasL number or higher coupling flexibility impede signaling. The results present DNA origami as versatile signaling scaffolds exhibiting unprecedented control over molecular number and geometry. They define molecular benchmarks in apoptosis signal initiation and constitute a new strategy to drive particular cell responses.

Lipid phase separation in vesicles enhances TRAIL-mediated cytotoxicity

“…Work studying Fas/FasL, which induces apoptosis similarly to TRAIL/DR5, demonstrated the ligand spacing of FasL affected the apoptosis rate, which could explain increased caspase activity in the phase segregated 2:1 and 3:1 DSPC:DOPC TRAIL compositions but not DOPC TRAIL. 27 These results confirmed the mode of TRAIL-vesicle mediated cytotoxicity was through caspase-mediated apoptosis, and that concentrating TRAIL into vesicle membrane domains increased caspase activity.…”

“…38 A similar study found precise ligand spatial orientation was critical for Fas (CD95) signaling, another TNF superfamily receptor with a similar signaling pathway as DR5. 27 We hypothesize that controlling TRAIL spatial orientation on lipid vesicles by using lipid domains most likely increases DR5-mediated apoptosis by approaching this critical inter-ligand distance.…”

Signal Amplification in Highly Ordered Networks Is Driven by Geometry

“…Using a DNA origami platform, the FasL ligands were immobilized and arranged in different geometries to test the effect of clustering on apoptosis efficiency in HeLa cells overexpressing the Fas receptor. Hexagonally arranged ligands signaled orders of magnitude better than soluble ligands [ 25 ]. Furthermore, ligands arranged in a hexagon with the right geometry doubled the rate of apoptosis compared to ligands arranged in pairs [ 25 ].…”

Strategies for the site-specific decoration of DNA origami nanostructures with functionally intact proteins

“…Instead, commercially available tetravalent streptavidin (tSAv) has often been used, and while it offers in principle three sites for ligand binding, only one may be accessible due to steric hindrance when surface-attached 33 . Differences in the cellular response to a ligand presented via mSAv and tSAv have been observed yet attributed to the presence of a flexible linker in the mSAv construct 7 . To assess biochemical and functional consequences of tSAv valency in more detail, we designed the construct H57-tSAv in analogy to H57-dSAv, but replaced the divalent with tetravalent streptavidin (SI Fig.…”

“…By using short oligonucleotides ("staples") to guide the folding of a long single stranded DNA scaffold, it is not only possible to program the shape of a DNA origami structure but also to arrange functional elements with nanometer resolution and precision 1 . Proteindecorated DNA origami structures have thus far been interfaced with cells as soluble agents 2,3 , attached to a solid substrate 4,5 or anchored to a fluid supported lipid bilayer (SLB) 6,7 ; biological questions addressed range from studying cellular signaling and adhesion processes [2][3][4][5][6]8,9 , to creating synthetic multienzyme cascades 10,11 to more and more elaborate robotic DNA machines 12 .…”