The Build of Functional Protein Molecular Shuttles by Using a Genetically Engineered Kinesin and Nucleic Acid Aptamers - with the Integration of the Cost-Effective Microscopic Imaging System and Micro Fluidic Channel Chips

Project: A - Government Institutionb - Ministry of Science and Technology

Description

The kinesin motors and microtubules are the intracellular transportation machinery of living cells, and they posses fascinating features which can be utilized to build the man-made molecular shuttles working in the micro fabricated devices. Microtubules (MTs) are protein tubes assembled with tubulin subunits. MTs are polarized in that the two ends (the – end and the + end) have different biochemical properties. Kinesin motors generally are made of two sets of heavy chains. The conventional kinesin proteins have three structural domains, and the motor domain is located at the N-terminal domain. The cargo-binding domain is at the tail (the C-terminal) while a coil-coil region in the middle. The interesting features of kinesin motors are that in the absence of cargo binding, the tails of kinesin fold back and prevent the motor domains to hydrolyze ATP and contact with MTs. Only upon the cargo loading, the kinesin motors can land on MTs and turn on the ATPase. In the living cells, by utilizing the energy from ATP hydrolysis the conventional kinesin motors carry specific organelles and bio-molecular complexes along the MTs from the - end to the + ends. To build functional kinesin shuttles operating in micro fluidic channels, three issues must be addressed. Firstly, how to let the shuttles, either the MTs or the kinesin, specifically carry the man-designated cargo molecules. Secondly, how to fabricate the micro fluidic channels and how to align the MT tracks so that the shuttles can move towards the same direction. Thirdly, how to observe the movements of the molecular shuttles. Although researchers have devoted lots of efforts, the practically useful protein shuttles remain to be realized. In this proposal, I use the recombinant protein approach to solve the problem of cargo reorganition and binding. I have genetically engineered a kinesin motor (KHCDB) and use it to carry a DNA aptamer. Aptamers are the man-screened single-stranded nucleic acids which can bind specifically with the designated targets. I have biochemically characterized the KHCDB, and observed the transportation of the KHCDB-bound DNA along MTs. Moreover, we have also very recently overcome the difficulties of microscope instrumentation and the fabrication of the micro fluidic channel chips. We are integrating the above three foundations to demonstrate the novelty and usefulness of this kinesin shuttle.
StatusFinished
Effective start/end date8/1/1210/31/13