Progress Towards Visualizing Single Molecules for Transcription Studies 公开
Eligulashvili, Anna (Spring 2022)
Published
Abstract
Deoxyribonucleic acid (DNA) is a complex biomolecule that serves as the universal genetic code and codes for all processes necessary for life. The first step in the expression of this genetic code is transcription, which is carried out by the ribonucleic acid polymerase (RNAP), a DNA-binding protein. In order to gain more insight on RNAP functional irregularities such as roadblocking and backtracking, various methods were explored for using the correlative optical trap (C-Trap) to obtain force, distance, and fluorescence data as a function of time. Using the high-resolution dual optical tweezers and multi-laser confocal microscopy system in the C-Trap, force-extension curves and kymographs were collected and analyzed for a variety of DNA tethers and fluorophores. Initial experiments aimed at visualizing the full length of a 14 kbp tether with SYBR Green DNA-intercalating dye. This experiment required high force to cause intercalation and molecules often ruptured. Other experiments were conducted with a 12 kbp DNA tether that was sparsely labeled with Cy5 fluorophores. While some data were promising, the resolution and lifetimes of the fluorophores were too low to be suitable in future transcription experiments. This drawback led to the use of an oxygen scavenger system consisting of a Glucose-Catalase-Oxidase oxygen scavenger and Trolox, a radical-reducing reagent, which together significantly increased the probability of DNA tethering and the lifetime of the fluorescent signal. In addition to visualizing DNA tethers, fluorescent experiments also confirmed the possibility of using labeled biotinylated RNAP labeled with Cy3-labeled streptavidin proteins to track RNAP movement along a tether. Preliminary experiments with RNAP showed specific binding of the enzyme to promoter regions, translocation, and disassociation. All fluorescent experiments were optimized by altering the concentrations of chemical reagents, employing oxygen scavenging methods, and adjusting the focus and filters of the confocal detectors. These aforementioned experiments show that the C-Trap can be used for such transcription experiments and provide protocols optimize the results.
Table of Contents
1 Introduction........................................................................................................................1
1.1 DNA as a Key Construct to Life ...........................................................................................1
1.2 DNA Mechanics.................................................................................................................2
1.3 RNA Polymerase and Transcription ....................................................................................4
1.4 Research Motivation .........................................................................................................6
1.5 Single Molecule Techniques in Biophysics...........................................................................7
2 The C-Trap Microscope ........................................................................................................10
2.1 Background ......................................................................................................................10
2.2 Microfluidics ....................................................................................................................11
2.3 Optical Tweezers ..............................................................................................................12
2.4 Fluorescence and its Use in a Model System ........................................................................13
2.5 Experimental Process and Optimization .............................................................................14
2.6 Advantages and Disadvantages ..........................................................................................16
3 Materials and Methods .........................................................................................................17
3.1 DNA Tether Construction with PCR ....................................................................................17
3.2 DNA Tether Construction with Labeled Nucleotides ............................................................18
3.3 DNA Tether Purification and Characterization with Gel Electrophoresis and AFM .................20
3.4 Sample and Flow Cell Preparation for C-Trap Use ...............................................................21
3.5 Tether Capturing and Manipulation in the C-Trap ..............................................................23
3.6 Confocal Microscopy with the C-Trap ................................................................................25
3.7 Oxygen Scavenger Methods for Improved Fluorescent Imaging ...........................................26
4 Results and Analysis ..........................................................................................................27
4.1 Force-Extension Curves Prove DNA Tethering ..................................................................27
4.2 Confocal Imaging with Fluorescent Intercalator Verifies DNA Tethering .............................29
4.3 Confocal Imaging of Fluorescently-Labeled DNA Verifies DNA Tethering ............................31
4.4 Confocal Microscopy Confirms RNAP Binding to DNA .......................................................34
4.5 Manipulation of Confocal Images for Optimized Fluorescent Visualization ............,,,,.........36
4.6 Troubleshooting .............................................................................................................38
5 Discussion and Conclusion ................................................................................................40
5.1 C-Trap Allows for Visualization of a Single DNA Tether and RNAP .....................................40
5.2 Optimization of Confocal Settings Improves Visualization of Fluorescent Signals ...............40
6 References.........................................................................................................................41
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