Powering Polyvalent DNA Motors Using Exonuclease III Open Access

Deng, Wenxiao (Spring 2021)

Permanent URL: https://etd.library.emory.edu/concern/etds/5999n476d?locale=en


Biological motor proteins have evolved the ability to efficiently convert the chemical energy stored in nucleotide triphosphates into mechanical work that powers locomotion and many other activities of living systems. Inspired by these biological motors, chemists and engineers have worked toward designing and assembling synthetic nano-scale machines that can convert chemical energy into controlled mechanical motion. Due to their predictability, DNA and RNA are more widely used in designing nanomotors compared to small molecules and other polymer materials. The motion of the vast majority of DNA-based motors is described by a burnt-bridge Brownian ratchet mechanism which is powered by the specific activity of enzymes. Nonetheless, DNA-based walkers fall short because of their limited speed, low endurance, and the lack of preferred directionality which is ultimately due to the lack of coordination between individual DNA legs. Standing out among the various DNA walkers is the DNA-based rolling motor that rolls on an RNA monolayer powered by RNase H. This class of DNA motor travels at an unprecedented speed with high processivity. However, the inherited instability of the RNA tracks limits this system from being applied in analytical sensing and in live cells. Therefore, it is highly desirable to design a DNA-based rolling motor that translocate on a DNA track via the rolling mechanism. In this thesis, we investigate an Exonuclease III-powered DNA motor that can roll on a DNA-based track without employing RNA fuel. We studied the specificity of Exonuclease III activity using DNA gold nanoparticle conjugates and fluorescence measurements in different enzyme conditions. Our investigations led to the design of motors that translocate 20 µm within 30-minute durations with a sub-population of motors exhibiting superdiffusive motion. The work points toward new synthetic motor systems that are more robust and that may one day allow for real-world applications of synthetic machines.  

Table of Contents

1. Introduction. 1

1.1 Background. 1

1.2 Aim and Scope. 7

2. Experimental Methods. 12

2.1 Preparation of gold surface. 13

2.1.1 Thermal evaporation of gold films. 13

2.1.2 Preparation of imaging chambers. 13

2.1.3 Fabrication of DNA monolayers on Au surface. 13

2.2 Fabrication of DNA monolayer on glass surface. 14

2.3 Synthesis of DNA-functionalized silica beads. 16

2.3.1 Synthesize of azide-functionalized particles. 16

2.3.2 Functionalization of DNA-coated silica particles. 16

2.4 Preparation of Rolling solution and imaging. 17

2.5 Trajectory Analysis. 17

2.6 Synthesis and characterization of DNA-functionalized Gold Nanoparticle. 18

2.6.1 Synthesis of DNA-functionalized particles. 18

2.6.2 Determining Gold Nanoparticle Loading Density. 18

2.6.3 Preparation of Gold Nanoparticle Solution & Plate reader Reading. 19

3. Results. 19

3.1 Gel electrophoresis. 24

3.2 Assembling DNA fuels on gold surface. 26

3.3 Assembling DNA fuels on glass surface. 28

3.4 Fluorescence-based kinetic assay for Exonuclease III activity. 34

3.4.1 Quantification of DNA loading density and quenching efficiency. 35

3.4.2 Specificity and reactivity of Exo III. 38

4. Conclusions. 43

5. References. 45

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