Assembling, Visualizing and Improving DNA Motors using Single Molecule Fluorescence

Abstract

A major obstacle in developing and improving complex DNA-machines, such as motors and robots, is the difficulty of characterizing the integrity, structural dynamics and function of the assembly intermediates and of the final product. Typically, non in-situ Gel and AFM and in-situ bulk fluorescence methods are used, but these methods often failed to provide detailed structural dynamic information sufficient for device rational improvements. We will present two different DNA-motors studied using single-molecule fluorescence techniques. One motor is made of DNA-track embedded in a DNA-origami on which bipedal DNA-walker is striding upon interacting with fuel/anti-fuel, while the second motor is design to adapt autonomous behavior. The single-molecule FRET method provides detailed information about the motors structure, and the ALEX, provides information about the motor integrity and helps cleaning the data, monitors the motor assembly process and demonstrates activity in real time. The binding rate of different fuel/anti-fuel type and sequence and the efficiency of their individual steps are compared to achieve maximum operation efficiency, speed, and reliability. The detailed structural dynamic information extracted allows deeper theoretical understanding of the system behavior, and as a consequence, leading to motor improvement.