To better understand the underlying principles by which biological motors operate, recent work has focused both on understanding their operational principles, and on designing new molecular motors ab initio. Here, by studying and designing motors which use Brownian motion and track asymmetry to bias the direction of motion, I gained insight into the underlying principles by which such motors operate. "Molecular spiders" [JACS. 128, 12693 (2006)] are one example of synthetic biomolecular walkers able to generate biased motion by coupling the chemical asymmetry arising from substrate binding and cleavage to bias their mechanical stepping.These DNA-based motors diffuse to their substrate track where productive binding between a molecular spider’s DNAzyme leg and a ssDNA substrate facilitates cleavage of the substrate. Once cleaved, the decreased binding affinity between the DNAzyme and resulting product allows the motor to diffuse along the track and form new interactions with uncleaved substrate molecules. Toinvestigate the origin of biased motion of molecular spiders, I have performed Monte Carlo simulations. Using my simulations, I also investigated their performance as molecular motors, and determined how to optimize their motor properties by modifying tunable experimental parameters in spider design. These studies assisted us in the design and construction of a novel protein-based synthetic motor, the "Lawnmower", which uses a burnt–bridges type of mechanism, the same as spiders, to autonomously and diffusively move forward. The lawnmower has trypsin proteases as blades, linked to a quantum dot hub, that interact with a one-dimensional peptide substrate track via binding to and cleavage of the substrates. Experimentally, it is confirmed with kinetic assays that ourlawnmower is an active motor and that there are an average number of 8 blades on each motor. I also outlined the synthesis and characterization of a highly modified DNA-peptide construct, which acts as the track for the lawnmower. For this, I employed PCR to generate a densely labeled DNA and click chemistry for peptide conjugation to the functionalized DNA. As an additional motors-relatedproject, I present the synthesis of a long one-dimensional DNA track with periodically repeating elements that provide specific binding sites for the "Tumbleweed" molecular motor.
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Thesis advisor: Forde, Nancy
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