Establishing kinetic activity of Exonuclease III on surface-immobilized substrates used in novel DNA rolling motors Open Access

Sheyitov, Bakai (Spring 2023)

Permanent URL:


The kinetic capabilities of the enzymes used in nanotechnology, including in molecular motors, are often overlooked and poorly defined. This study aims to investigate the kinetic and biophysical profile of Exonuclease III, a common 3'-to-5' dsDNA exonuclease, and find its cleavage rate of two types of DNA substrates: freely floating in solution and immobilized on the surface. Comparing the hydrolysis rates in two environments allowed us to draw conclusions about Exonuclease III's kinetic activity and potential use in creating synthetic motors, specifically rolling motors. The rolling motor is a design in which the enzyme consumption of the nucleic acid monolayer propels the super-diffusive motion of the particle with multivalent DNA legs. An earlier generation of motors used RNase H hydrolysis of the RNA monolayer to power such motion. This work focuses on applying the findings from in-solution and on-surface kinetic assays to optimization of the design of RNA-independent rolling nanomotors. Our results show that Exonuclease III is capable of processive cleavage of DNA substrates in both conditions with in-solution kcat of 12.8 min-1, on-surface kcat of 0.1-0.3 min-1, and on-surface koff of 0.1-0.2 min-1 when 8 nM ExoIII was added to the buffer containing 10 mM of Mg2+. Additionally, the ability of certain protecting groups and fluorophores to affect the enzymatic cleavage was tested. A closer look at the Exonuclease III's behavior in various conditions and with substrates in distinct biophysical settings (in solution and on surface) addressed the fundamental questions of enzyme kinetics and laid a foundation for engineering a new class of nucleic acid-based molecular motors.

Table of Contents

Background 1

Methods 24

Results and Discussion 31

Conclusion and Future Directions 54

References 55

About this Honors Thesis

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
  • English
Research Field
Committee Chair / Thesis Advisor
Last modified

Primary PDF

Supplemental Files