Abstract
Fluorescence-labeled tiny DNA origami blocks (FTOBs) enable observation of kinesin motility and their cooperation with minimal blinking and photobleaching. Here, we present a protocol for assembling FTOBs to observe kinesin cooperation. We describe steps for linking FTOBs and kinesins via a rationally designed epitope tag (ALFA-tag) and its high-affinity single-domain nanobody (NbALFA). We detail procedures for connecting two FTOB-kinesin complexes via hybridization of complementary DNA arms and their single-molecule tracking. This protocol has potential applications in any motor proteins. For complete details on the use and execution of this protocol, please refer to Kita et al.1.