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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 • Preparation of a photostable fluorescence-labeled tiny DNA origami block (FTOB) • Steps for attaching nanobody-fused kinesin on FTOB using the ALFA-tag • Instructions for linking two FTOB-kinesin complexes via hybridization • FTOB enables the observation of collective motion of kinesins using TIRF microscopy Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Kita et al. (Mon,) studied this question.