Modular photostable fluorescent DNA blocks dissect the effects of pathogenic mutant kinesin on collective transport

Intracellular transport is driven by teams of various motor proteins. Advances in DNA nanotechnology have enabled the programmable linkage of different types of motor proteins. In this study, we developed a modular, photostable fluorescence-labeled tiny DNA origami block (FTOB) for extended observation of collective transport by selected motor combinations. The FTOB is designed as a 4-helix bundle (∼8.8 nm) with densely accumulated fluorescent dyes, minimizing blinking and photobleaching. By designing a pair of connector DNAs, FTOBs are heterodimerized following motor protein attachment using the ALFA-tag/nanobody system. Our system examined the impact of a pathogenic mutant kinesin on its collective movement with wild-type kinesin, clearly observing two distinct behaviors: the team’s velocity was generally governed by the slower mutant but occasionally surged to levels comparable to that of a single wild-type motor. Our photostable, robust, modular FTOB system could serve as a versatile tool for precisely dissecting cooperative cargo transport. • A fluorescence-labeled tiny DNA origami block (FTOB) showing high photostability is presented • FTOB has structural flexibility and can be dimerized by hybridization • Proteins of interest can be effectively linked to FTOB via ALFA-tag/nanobody system • FTOB gives insights into the inhibitory mechanism of a pathogenic mutant kinesin Blinking and photobleaching limit the tracking of molecules in single-molecule assays. Kita et al. develop modular, photostable fluorescence-labeled tiny DNA origami blocks (FTOBs) that can attach proteins of interest via the ALFA-tag/nanobody system. FTOB shows the inhibitory and non-inhibitory modes of a disease-associated mutant kinesin on collective transport.