Microtubule-kinesin system is considered as a building block for the construction of artificial biomachines, and active selforganization of microtubules has been used to integrate their structural organization and achieve amplified functions similar to those observed in natural systems. However, the short lifetime of assembled structures has limited their use in organized systems. In the present study, we demonstrated that the use of an inert atmosphere in the self-organization of microtubules allows the assembled structures to remain active for a prolonged period of time (10 times longer). The longer lifetime achieved in the present study will facilitate the development of assembled microtubules for designing biomolecular motor-based efficient artificial biomachines with prolonged lifetimes. Polymer Journal ( Keywords: active self-organization; biomachine; biomolecular motor; kinesin; lifetime; microtubule INTRODUCTION Actin-myosin and microtubule (MT)-kinesin systems are known for their fascinating in vivo activities with respect to cell motility, 1 cytokinesis 2 and cellular transport. To unveil the functions of bimolecular motor systems 3-5 and to elucidate the in vivo mechanism of actin-myosin or MT-kinesin interactions, in vitro motility assay 6,7 has been used over the last few decades. Currently, the in vitro motility assay is also used to determine the feasibility of constructing motor protein-based artificial biomachines, for which actin-myosin or MT-kinesin systems are considered promising building blocks. [8][9][10][11][12][13][14][15][16] New classes of devices including nanoscale molecular shuttles, 17,18 surface-imaging processes, 19 force measurements 20 and lab-on-a-chip devices 21 have been developed using knowledge obtained through the in vitro motility assay. To improve the organizational hierarchy of motor protein-based systems with emergent functions similar to those observed in natural systems, several active self-organization (AcSO) techniques have been developed. [22][23][24][25] For instance, a specific streptavidin (St)-biotin (Bt) interaction has been used in the in vitro motility assay of MTs on a kinesin-fixed surface, and a variety of well-organized MTs assemblies have been produced including linear bundles, rings and network structures, all of which showed motility comparable to that of single MT filaments. However, the lifetime of biomolecular motors is short owing to the attack of reactive oxygen species (ROS), which terminates their activity and limits the long-term utilization of assembled structures. Thus, assembled structures formed under ambient aerobic conditions remain active for only B90 min.