Proximity-induced caspase-9 activation on a DNA origami-based synthetic apoptosome

Abstract

Living cells regulate key cellular processes by spatial organisation of catalytically active proteins in higher-order signalling complexes. These act as organising centres to facilitate proximityinduced activation and inhibition of multiple intrinsically weakly associating signalling components, which makes elucidation of the underlying protein-protein interactions challenging. Here we show that DNA origami nanostructures provide a programmable molecular platform for the systematic analysis of signalling proteins by engineering a synthetic DNA origami-based version of the apoptosome, a multi-protein complex that regulates apoptosis by co-localizing multiple caspase-9 monomers. Tethering of both wildtype and inactive caspase-9 variants to a DNA origami platform demonstrates that enzymatic activity is induced by proximity-driven dimerization with half-of-sites reactivity, and additionally, reveals a multivalent activity enhancement in oligomers of three and four enzymes. Our results offer fundamental insights in Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Determinants of ligand-functionalized DNA nanostructure-cell interactions

“…Encouraged by the observed relation between DNA nanorod orientation and cellular binding efficiency, we constructed multiple DNA nanostructures to evaluate the effect of shape and size on receptor binding. Two additional nanostructures, a twist-corrected rectangular DNA origami rectangle 20,49 (Rec, 75×100 nm 2 ) and a tetrahedral DNA nanostructure 50 (Tet), respectively, were successfully folded and purified ( Fig. 3a and Supplementary Fig.…”

Challenges and Perspectives of DNA Nanostructures in Biomedicine

“…[26,36].Itisnoteworthy that harnessing DNAtemplates forprotein assembly mayhaveratherintriguinga nd unconventional implementationsi nt ailoredp rotein design,a sd emonstratedb yR osiere tal. [37] They assembled af unctionala poptosomeb yc o-localization of multiple caspase-9 monomers with theh elpo faDO platform. This approach may pave thew ay fort he engineeringo fa rtificial enzymest hata re involved in processess ucha si nflammation, innate immunity,and necrosis.…”

“…. It is noteworthy that harnessing DNA templates for protein assembly may have rather intriguing and unconventional implementations in tailored protein design, as demonstrated by Rosier et al . They assembled a functional apoptosome by co‐localization of multiple caspase‐9 monomers with the help of a DO platform.…”

DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation

“…Staple strands may also serve as handles for attaching diverse molecular components to enable novel types of biological experiments. We and others have used DNA origami to stimulate cells via patterned display of antibody fragments, peptides, or native ligands (Douglas et al, 2012; Hellmeier et al, 2021; Huang et al, 2019; Rosier et al, 2020; Shaw et al, 2019; Veneziano et al, 2020). However, relying on protein-based ligands introduces additional technical challenges, including incomplete functionalization (Hellmeier et al, 2021), and fine-tuning the strength and conformational preference of receptor-ligand interactions (Shaw et al, 2019).…”