Protocol for assembling photostable fluorescence-labeled tiny DNA origami blocks to observe kinesin cooperation
Summary: 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...
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| Language: | English |
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Elsevier
2025-09-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725003727 |
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| author | Tomoki Kita Ryota Sugie Yuki Suzuki Shinsuke Niwa |
| author_facet | Tomoki Kita Ryota Sugie Yuki Suzuki Shinsuke Niwa |
| author_sort | Tomoki Kita |
| collection | DOAJ |
| description | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-74b0d8b2918f4c91816f6953eccf2b5e |
| institution | Kabale University |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-74b0d8b2918f4c91816f6953eccf2b5e2025-08-20T03:25:49ZengElsevierSTAR Protocols2666-16672025-09-016310396610.1016/j.xpro.2025.103966Protocol for assembling photostable fluorescence-labeled tiny DNA origami blocks to observe kinesin cooperationTomoki Kita0Ryota Sugie1Yuki Suzuki2Shinsuke Niwa3Graduate School of Life Sciences, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai, Miyagi 980-8577, JapanDepartment of Applied Chemistry, Graduate School of Engineering, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, JapanDepartment of Applied Chemistry, Graduate School of Engineering, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan; Corresponding authorGraduate School of Life Sciences, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai, Miyagi 980-8577, Japan; Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku University, Aramaki-Aoba 6-3, Aoba-ku, Sendai, Miyagi 980-0845, Japan; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166725003727BiophysicsSingle-molecule AssaysMicroscopyMolecular BiologyMolecular/Chemical ProbesProtein Biochemistry |
| spellingShingle | Tomoki Kita Ryota Sugie Yuki Suzuki Shinsuke Niwa Protocol for assembling photostable fluorescence-labeled tiny DNA origami blocks to observe kinesin cooperation STAR Protocols Biophysics Single-molecule Assays Microscopy Molecular Biology Molecular/Chemical Probes Protein Biochemistry |
| title | Protocol for assembling photostable fluorescence-labeled tiny DNA origami blocks to observe kinesin cooperation |
| title_full | Protocol for assembling photostable fluorescence-labeled tiny DNA origami blocks to observe kinesin cooperation |
| title_fullStr | Protocol for assembling photostable fluorescence-labeled tiny DNA origami blocks to observe kinesin cooperation |
| title_full_unstemmed | Protocol for assembling photostable fluorescence-labeled tiny DNA origami blocks to observe kinesin cooperation |
| title_short | Protocol for assembling photostable fluorescence-labeled tiny DNA origami blocks to observe kinesin cooperation |
| title_sort | protocol for assembling photostable fluorescence labeled tiny dna origami blocks to observe kinesin cooperation |
| topic | Biophysics Single-molecule Assays Microscopy Molecular Biology Molecular/Chemical Probes Protein Biochemistry |
| url | http://www.sciencedirect.com/science/article/pii/S2666166725003727 |
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