Programmable DNA-peptide nanostructures for multivalent regulation of intracellular signalling

The Wnt/{beta}-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 {beta}-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.