The Use of Nucleic Acid Aptamer to Build Kinesin Molecular Shuttles

To bring about the further advancement of technologies using miniature machines, the importance of design and synthesis of molecular motors has been recognized in the award of Nobel Prize in Chemistry 2016. In fact, in the biological world, protein motors have demonstrated the benefits of using them in dealing with the issues of separation and transportation. Inspired by the studies of protein kinesin motors and microtubules (MTs), researchers have proposed the build of molecular shuttles with kinesin and MTs. Kinesin motors transport specific cargos (e.g., native organelles and macro molecular complexes) along MTs by using energy stored in ATP. Notably, individually kinesin motors intrinsically have low processivity (run-length) and tend to fall off from MTs after ~125 steps (or ~1 m). To carry cargos across eukaryotic cells, kinesin motors overcome the issues of low processivity by collectively transport cargos. Researchers have made two types of shuttles, MT shuttles and kinesin shuttle. MT shuttles can transport cargo for very long distances. Although most researchers favor the model of MT shuttles, the progress of making useful MT shuttles have almost stopped in recent years, due to the inborn shortcomings of MT shuttles. On the other hand, although the use of kinesin shuttles is more promising, we need to solve the issue of cargo-loading and collective transportations. To pursuit the build of useful kinesin shuttle, I proposed the use of cargo-binding aptamers, scaffold DNA, and an engineered kinesin, KHC-Cro protein motor. KHC-Cro motors contain DNA-binding domains, and, as expected, they specifically transported double-stranded scaffold DNA (dsDNA) with Cro-binding sequences (Current Nanoscience, 2012. 8(5): 669-674). Also we have built an imaging system with a DIC microscope, sensitive camera and imaging processing algorithm. A method of making simple microfluidic devices is established. Furthermore, we have observed the collective transportation of streptavidin (SA) coated-latex beads by KHC-Cro shuttles. The binding between the beads and kinesin is mediated by a SA-binding aptamer in a noncovalent manner. The aptamer can be easily changed for kinesin to carry different target cargos. This approach is very different from the ways of cargo-loading of MT shuttles where chemical cross-linkages are used to tether cargo-adaptor to MT shuttles. The aims of this 2-year proposal are to accomplish the following 2 tasks, I. To characterize and improve the binding and transport of target protein by aptamer-tagged collective kinesin motors (2017~2018). II. Exploit the collective transports of aptamer-tagged kinesin shuttles to detect the concentration of the interested target proteins (2018-2019). The feasibility of this proposal can be seen form the results of our preliminary studies. We have identify crucial issues which inhibit aptamers to bind their target cargos. We also proposed the use of SELEX protocol to screen for modified aptamers to rescue the target binding. Second, we have prepared DNA origami as a scaffold, which will be used with split aptamer and kinesin shuttles, to have cargo-promoted collective transportation of kinesin motors.