EVIDENCE FOR DNA OXIDATION IN SINGLE MOLECULE FLUORESCENCE STUDIES A thesis presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Master of Science Douglas Wylie August 2006 This thesis entitled EVIDENCE FOR DNA OXIDATION IN SINGLE MOLECULE FLUORESCENCE STUDIES by DOUGLAS WYLIE has been approved for the Department of Physics and Astronomy and the College of Arts and Sciences by Ido Braslavsky Assistant Professor of Physics and Astronomy Benjamin M. Ogles Dean, College of Arts and Sciences WYLIE, DOUGLAS, M.S., August 2006. Physics and Astronomy EVIDENCE FOR DNA OXIDATION IN SINGLE MOLECULE FLUORESCENCE STUDIES (103 pp.) Director of Thesis: Ido Braslavsky Single molecule fluorescence microscopy is a powerful tool to investigate local environments at the nanometer scale. Oxidation of DNA is an important problem that leads to DNA mutations. In this project, single molecule fluorescence measurements were used to study the interaction between covalently bound nucleotides (dNTP) and fluorophores (Cyanine dye), dNTP-Cy3 that have been incorporated into DNA. By tracing changes in the fluorescence signal, evidence for single events of DNA oxidation were found that could otherwise not be observed at the ensemble level. The differences between the nucleotides and their quenching properties are shown here especially guanine’s ability to temporarily quench the fluorescence of Cy3 completely. After some illumination time, the quenching of the dye by the guanine changes dramatically. We interpret this change to possible oxidation of the guanine base. This work could lead to a method for monitoring and investigating important DNA oxidation processes which are essential to Genomic and Cancer research. Approved: Ido Braslavsky Assistant Professor of Physics and Astronomy 4 Table of Contents Page Abstract......................................................................................................................3 List of Figures ............................................................................................................6 1 Introduction and Theory............................................................................................8 1.1 Importance of understanding DNA damage............................................................9 1.1.1 Single Nucleotide Polymorphisms (SNPs).....................................................10 1.2 Causes of DNA damage and their effects .............................................................12 1.2.1 Radicals ........................................................................................................12 1.2.2 Oxygen and its reactive derivatives, ROS......................................................14 1.3 One-electron oxidation of DNA with emphasis on guanine ..................................16 1.3.1 Observation of site-selective Guanine-Oxidation...........................................19 1.4 What is fluorescence? ..........................................................................................21 1.4.1 History of fluorescence .................................................................................21 1.4.2 Stokes’ shift phenomenon .............................................................................22 1.4.3 Jablonski energy diagram and quenching pathways .......................................24 1.4.4 Photobleaching and triplet-state reactions......................................................26 1.5 Total Internal Reflection Fluorescent Microscopy (TIRFM).................................31 1.6 Brief experimental outline and aim ......................................................................34 2 Sample Preparation and Experimental Method......................................................35 2.1 Laser setup and TIR illumination .........................................................................35 2.2 Cleaning procedures and surface chemistry protocols ..........................................39 2.2.1 Cleaning the glass slides................................................................................40 2.2.2 Application of polyelectrolyte multilayers.....................................................41 2.2.3 Surface activation for further attachments......................................................42 2.2.4 Streptavidin application procedure ................................................................43 2.2.5 Preparation and application of washing buffers..............................................45 2.3 Properties of Cyanine Dyes, and DNA preparation...............................................45 2.4 Observation of sample and data acquisition..........................................................51 2.5 Initial data analysis using custom developed correlation software ........................54 3 Results and Comments .............................................................................................56 3.1 Editing and analysis of movie data.......................................................................56 3.2 Cy3-Primer/DNA by itself. ..................................................................................58 3.3 dATP-Cy3/DNA..................................................................................................61 3.4 dCTP-Cy3/DNA. .................................................................................................64 3.5 dUTP-Cy3-Primer/DNA (T404)...........................................................................66 3.5.1 dUTP-Cy3/DNA (T407)................................................................................70 3.6 dGTP-Cy3/DNA..................................................................................................74 3.6.1 dGTP-Cy3/DNA (sample 2)..........................................................................77 3.7 Preliminary results for incorporated dGTP-Cy5 with oxygen scavenger solution..80 5 Page 4 Discussion..................................................................................................................83 4.1 Evidence for Guanine oxidation...........................................................................83 4.2 New light on interpretation of photobleaching kinetics in single-molecule fluorescent traces. ......................................................................................................91 5 Conclusions and Suggestions for Future Work.......................................................92 6 References.................................................................................................................95 6 List of Figures Page Figure 1) Ball and stick image of DNA double-helix.......................................................8 Figure 2) Chemical Structure of all 4 DNA bases along with Uracil present in RNA instead of Thymine...............................................................................10 Figure 3) Diagram showing the π - stacking interactions among intrastrand bases and also hydrogen bonding between complementary base-pairs.....................14 Figure 4) An example of how oxidation of the original guanine base can induce mis-pairing of complementary bases..............................................................18 Figure 5) Structure of Cy3 (top) covalently attached to the de-oxy Cytosine Triphosphate nucleotide, dCTP (bottom).......................................................20 Figure 6) Typical diagram showing Stokes’ shifted absorption and emission spectra...........................................................................................................22 Figure 7) Diagram showing lowering of excited state singlet levels of a polar fluorophore due to solvent relaxation with surrounding medium....................24 Figure 8) Jablonski diagram showing possible pathways for an excited electron and the various energy losses, transfer mechanisms and timescales for each transition ......................................................................................................25 Figure 9) Graph showing fluorescent intensity vs time for 3 single-molecules of Cy3 attached to a DNA-Primer sequence...................................................27 Figure 10) Simplified Jablonski diagram showing various reaction rates and photobleaching behavior .......................................................................29 Figure 11) Picture showing path taken by laser light through objective and reflected at cover glass/sample surface interface ........................................................32 Figure 12) Nice diagram depicting approximate penetration depth d, of the evanescent wave at an incident angle of 62 degrees for a 542nm wavelength laser beam…. .......................................................................................................33 Figure 13) Microscope with red (top left) and green (middle left) laser set-up on air-cushioned table ......................................................................................35 Figure 14 a and b) Absorbance and emission spectral profiles for the cyanine dyes, Cy3 and Cy5, along with their appropriate filter cubes allowing maximum desired light through with minimal overlap of unwanted light ......................36 Figure 15) Plot showing relative intensity of evanescent wave against displacement from surface, z (nm) and angle of incidence, θ (degrees) for 532nm wavelength excitation 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