P09-01
Modular photostable fluorescent DNA blocks for tracking collective movements of motor proteins
Ryota SUGIE *1, Tomoki KITA2, Shinsuke NIWA2, 3, Yuki SUZUKI1
1Department of Applied Chemistry, Graduate School of Engineering, Mie University
2Graduate school of Life Sciences, Tohoku University
3Frontier Research Institute for Interdisciplinary Sciences(FRIS), Tohoku University
( * E-mail: 423m328@m.mie-u.ac.jp )
Active intracellular transport is carried out by the collective action of multiple motor proteins. The properties of this process depend not only on the number of motor proteins responsible for transporting the cargo but also on the specific types of motor proteins involved. DNA nanotechnology has provided an attractive approach for modeling such a complex system by allowing different types of motors to be linked in a programmable manner. The movements of the assembled complex are often tracked using total internal reflection fluorescence (TIRF) microscopy; however, the blinking and photobleaching of fluorescent dyes limit the duration of imaging, which in turn restricts detailed analysis of the collective movements. In this study, we developed a connectable photostable DNA nanostructure, designated as the fluorescence-labeled tiny DNA origami block (FTOB). The FTOB is a 4-helix bundle of approximately 8.4 nm in size, in which five (or six) fluorescent dyes have been integrated, resulting in its minimal blinking and photobleaching properties. By designing a pair of connector DNAs, FTOB can be heterodimerized after the attachment of the motor protein of interest via the ALFA-tag/NbALFA system, thereby enabling the formation of a mimic of a cargo transport complex with a selected combination of motor proteins. We believe that our modular FTOB system would serve as a novel tool for the investigation of cooperative cargo transport at the single molecule level.