A semisynthetic protease DNA-nanomachine
Final Report Abstract
The proposed project aims at the construction of a DNA-scaffolded protein degradation nanomachine. First, desired enzymes are confined within DNA origami compartments, with control over the number of bound enzyme molecules and their relative positions. Then, a limited number of those compartments are linked together in a lane, with each compartment loaded with a distinct enzyme. The final construct is a nanochannel composed of multiple and connected compartments capable to perform distinct and spatially localized biochemical reactions on a given substrate. The next level of sophistication is the introduction of a lid at one side of the channel to control the entry of the substrate and its progressive enzymatic processing throughout the entire cascade. This will allow to perform desired biochemical reactions with control over their spatial location and temporal order. The initial idea was to use three serine proteases in a lane to mimic the structural principles and proteolytic activity of the proteasome. This system however typically requires misfolded or unfolded proteins to be really effective. We therefore introduced a segregase/unfoldase p97 machine at the entry of the nanochannel to extend the application of our system to compact protein substrates even in complex with other proteins. The DNA-encaged p97 retains full activity and the protein pore is aligned along the central axis of the cavity, thus enabling downstream proteolysis of the unfolded substrate in the adjacent chymotrypsin-loaded chamber. The two-chambers DNA nanochannel is therefore capable to perform unfolding and proteolysis of a substrate in a programmable sequential order. This new direction of the project notably increased the load and challenge of the work but enabled to emulate a more sophisticated cellular process and most importantly holds the potential to achieve more ambitious goals in the future. We are confident that our findings will be of interest for the scientific community and may stimulate further research in the field of synthetic biology and nanomaterial science.
Publications
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Manipulating Enzymes Properties with DNA Nanostructures. Molecules 2019, 24(20), 3694
Jaekel A, Stegemann P, Saccà B
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Sites of high local frustration in DNA origami. Nat. Commun. 2019, 10(1):1061
Kosinski R, Mukhortava A, Pfeifer W, Candelli A, Rauch P, Saccà B
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Site-specific facet protection of gold nanoparticles inside a 3D DNA origami box: a tool for molecular plasmonics. Chem. Commun. 2021, 57(25), 3151-3153
Erkelenz M, Kosinski R, Sritharan O, Giesler H, Saccà B, Schlücker S
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Self-Assembled Artificial DNA Nanocompartments and Their Bioapplications. Small 2022, Jul 1:e2202253
Huang J, Gambietz S, Saccà B
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The role of DNA nanostructures in the catalytic properties of an allosterically regulated protease. Sci. Adv. 2022, 8(1), eabk0425
Kosinski R, Mieres Perez J, Schöneweiß EC, Ruiz-Blanco YB, Ponzo I, Bravo-Rodriguez K, Erkelenz M, Schlücker S, Uhlenbrock G, Sanchez-Garcia E, Saccà B
