Programmable DNA-peptide nanostructures for multivalent regulation of intracellular signalling

The Wnt/β-catenin pathway is constitutively active in most colorectal and other cancers. Tankyrase (TNKS) promotes Wnt signalling by PARylating AXIN, a rate-limiting scaffold subunit of the β-catenin destruction complex, and its inhibition is a validated strategy for pathway downregulation. Existing small-molecule TNKS inhibitors that target the catalytic PARP domain suffer from off-target effects across the PARP family. Here, we present an alternative approach that targets the substrate-binding domains of TNKS using a short TNKS-binding peptide (TBP) presented multivalently on DNA nanostructures. Such a strategy may be required to effectively disrupt the function of targets such as TNKS, which is known to form high-order assemblies in the cell. We engineered DNA nanostructures with two distinct geometries: a 2D triangle (∼100 nm) displaying 27 copies of TBP, and a compact 3D tetrahedron (∼10 nm) displaying 2 TBP copies. We show that both nanostructures assemble efficiently, can be functionalised with TBP in high yields, and retain binding to TNKS protein in vitro . DNA nanostructures are efficiently internalised to the cytoplasm by HeLa Kyoto and colorectal cancer cells, with TBP functionalisation enhancing rather than hindering uptake. In HeLa cells, both triangle-TBP and tetrahedron-TBP downregulated Wnt signalling to a similar extent despite an order-of-magnitude difference in TBP copy number, while the same concentration of free TBP had no effect. This finding likely reflects the two functions of the DNA nanostructures - intracellular delivery and multivalent display - whereby the smaller nanostructures more efficiently internalise the TBP ligand but have lower valency. These results establish DNA nanostructures as a modular, tuneable platform for multivalent inhibition of intracellular clustered targets such as TNKS.