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Single-Molecule Biophysics of Dna Bending: Looping and Unlooping

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Single-Molecule Biophysics of Dna Bending: Looping and Unlooping SINGLE-MOLECULE BIOPHYSICS OF DNA BENDING: LOOPING AND UNLOOPING A Dissertation Presented to The Academic Faculty by Tung T. Le In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy in the School of Physics Georgia Institute of Technology August 2015 Copyright © Tung T. Le 2015 SINGLE-MOLECULE BIOPHYSICS OF DNA BENDING: LOOPING AND UNLOOPING Approved by: Professor Harold Kim, Advisor Professor James Gumbart School of Physics School of Physics Georgia Institute of Technology Georgia Institute of Technology Professor Daniel Goldman Professor Loren Williams School of Physics School of Chemistry and Biochemistry Georgia Institute of Technology Georgia Institute of Technology Professor Jennifer Curtis Date Approved: April 09, 2015 School of Physics Georgia Institute of Technology A dedication to my father, with love. ACKNOWLEDGEMENTS First, I would like to give my highest appreciation to my advisor, Dr. Harold D. Kim, for his enduring guidance and generous support to my research during the graduate years. He has given me a unique opportunity to work independently and judge things in a scientific manner. I would like to thank my previous advisor, Dr. Toan Nguyen, for his great mentor and support during my first year at Georgia Tech. I would also like to extend my appreciation to my committee members: Dr. Daniel Goldman, Dr. Jennifer Curtis, Dr. James Gumbart, and Dr. Loren Williams, for reading my thesis and giving me valuable comments. I want to thank Dr. Rasesh Parikh and other members of the Kim's lab for their kindness. Outside the lab, I would like to give thanks to many people, my dear friends (Phuong Doan, Tien Hoang, Phuong Hoang) and many others in the Vietnamese Student Associa- tions (VSA) in Atlanta, and my close friends at home (Vuong Linh Tran, Duy Nguyen) for their love and friendship. Finally, I would like to express my deepest gratitude to my beloved parents and my two sisters. I want to honor my father, who has recently passed away, for having strong beliefs in me and in my choice. I really want him to feel so proud of me. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................. iv LIST OF TABLES ................................... viii LIST OF FIGURES .................................. ix SUMMARY ........................................ xi I INTRODUCTION ................................. 1 1.1 DNA bends . .1 1.2 The worm-like chain model of DNA . .3 1.3 J factor . .6 1.4 Battles for and against the WLC model . .8 1.5 Reaching a unified view of strong DNA bending . 10 1.6 Structure of the thesis . 12 II SINGLE-MOLECULE FLUORESCENCE RESONANCE ENERGY TRANS- FER AND TOTAL INTERNAL REFLECTION FLUORESCENCE MI- CROSCOPY ..................................... 13 2.1 Fluorescence resonance energy transfer (FRET) . 13 2.2 Experimental design . 17 2.2.1 Single-molecule fluorescence dyes . 17 2.2.2 Enhancing photophysical stability . 19 2.3 Single molecule detection . 20 2.3.1 Total internal reflection and the objective-type total internal reflec- tion microscopy . 20 2.3.2 Objective-type TIRFM setup . 23 2.3.3 Emission detection for dual-view imaging . 23 2.3.4 Temperature control of the sample via the objective . 25 2.4 Sample preparation to data acquisition . 25 2.4.1 Surface immobilization . 25 2.4.2 Flow cell preparation . 28 2.5 Data processing and analysis . 29 2.5.1 Data processing . 29 v 2.5.2 Data analysis . 30 III SINGLE-MOLECULE LOOPING ASSAY OF SHORT DOUBLE-STRANDED DNA TO STUDY THE EFFECT OF SEQUENCE CURVATURE ON DNA LOOPING .................................. 35 3.1 Introduction . 36 3.2 Experimental Design . 37 3.2.1 186-bp long synthetic DNA sequences with varying shape (for each sequence, one repeating unit is put in the parentheses) . 38 3.2.2 Genomic DNA sequences with varying length . 39 3.2.3 Making the dsDNA with fluorophore and biotin label using PCR . 39 3.2.4 DNA electrophoresis . 40 3.2.5 Image acquisition . 41 3.2.6 Data analysis . 42 3.3 Measuring DNA looping dynamics . 44 3.3.1 FRET fluctuations reflect looping dynamics of dsDNA . 44 3.3.2 Conversion of jM from looping rate . 47 3.3.3 More curved molecules loop faster . 49 3.4 Coarse-grained simulations . 51 3.4.1 The wedge-angle model for dsDNA deformation . 51 3.4.2 Adding the thermal fluctuations . 54 3.4.3 Numerical prediction of the J factor . 55 3.4.4 Optimizing helical parameters from gel mobility . 57 3.5 Comparison of experiment with WLC simulation . 59 3.5.1 The effect of intrinsic curvature on the looping probability of DNA sequence . 59 3.5.2 Looping probability of a genomic sequence DNA as a function of length . 61 3.6 Discussion . 62 IV UNLOOPING ASSAY TO TO INVESTIGATE THE BENDING ME- CHANICS OF DNA UNDER STRONG BENDING CONDITIONS . 65 4.1 Introduction . 66 4.2 Shear force and loop breakage dynamics . 68 4.3 Experimental design and methods . 70 vi 4.3.1 Materials . 70 4.3.2 Single-molecule unlooping assay . 72 4.4 Smaller DNA loops are less stable . 74 4.5 Shear force calculations . 74 4.5.1 DNA models and parameters . 74 4.5.2 Monte Carlo simulation for dsDNA bending with Metropolis criterion 76 4.5.3 Biased Monte Carlo simulation for simulating small DNA loops . 77 4.5.4 Shear force for DNA loops with varying size . 79 4.6 Analysis of the linker lifetime vs. shear force reveals softening transition of a worm-like chain . 81 4.7 Softening transition is sensitive to magnesium . 85 4.8 Discussion . 87 V FUTURE PROSPECTS - SEQUENCE DEPENDENT KINKABILITY 93 5.1 Introduction . 93 5.2 Methods . 94 5.3 Preliminary Results . 97 5.3.1 Looping dynamics of some DNA sequences with varying nucleosome affinity . 97 5.3.2 Looping dynamics as a function of GC percentage . 99 5.3.3 Some DNAs with repeating patterns show enhanced flexibility . 100 5.4 Final remarks . 101 APPENDIX A | SUPPLEMENTARY METHODS FOR CHAPTER IV103 APPENDIX B | SUPPLEMENTARY METHODS FOR CHAPTER V 105 APPENDIX C | PERMISSIONS TO REUSE PREVIOUSLY PUBLISHED MATERIALS .................................... 107 REFERENCES ..................................... 108 VITA ............................................ 119 vii LIST OF TABLES 2.1 List of optics for single-molecule FRET setup using TIRFM and reagents for the DNA looping and unlooping assays . 31 3.1 The apparent size of several 186bp DNA sequences determined from a 100bp DNA marker. 50 3.2 The helical parameters for some dinucleotide steps from the Bolshoy-Trifonov model....................................... 53 3.3 The variances for the angle fluctuations of some dinucleotide steps from the Olson-Zhurkin model . 56 viii LIST OF FIGURES 1.1 The double-stranded DNA structure and DNA bending in the nucleosome .1 1.2 Freely rotating chain model . .3 1.3 Theoretical J factor predicted from the WLC model . .7 1.4 Schematic representation of cyclization . .8 2.1 FRET efficiency EFRET(R)........................... 14 2.2 Time-resolved FRET trace and FRET histogram . 16 2.3 Fluorescence Cy3 and Cy5, their absorbance and emisison spectrum . 18 2.4 Effects of the oxygen scavenging system and Trolox on the photophysics of single DNA-conjugated Cy5 molecules . 19 2.5 Objective-type TIR . 21 2.6 Characteristic penetration of the evanescent field . 22 2.7 Setup for objective-type TIRFM . 24 2.8 Temperature control for single-molecule experiments . 26 2.9 Passivation methods using Biotin-BSA and PEG . 27 2.10 Flow cell assembly . 28 2.11 GUI MATLAB program for data analysis . 30 3.1 Curved dsDNA . 37 3.2 PCR-based preparation of dsDNA . 41 3.3 A schematic of smFRET DNA looping assay and a typical time-resolved trace of Cy3 and Cy5 intensities . 43 3.4 Effect of the annealing overhangs on the DNA unlooping dynamics . 44 3.5 Salt dependence of looped and unlooped state lifetimes . 45 3.6 Effect of sequence length on the looping kinetics . 46 3.7 Dependence of the apparent looping kinetics on the acquisition time . 47 3.8 Determining the annealing rate constant . 49 3.9 Electrophorentic mobility of several curved DNAs and their looping kinetics 50 3.10 Orientational parameters of a base pair plane in a dinucleotide step . 52 3.11 Defining base-pair coordinates . 54 3.12 Determing the looping jM ........................... 56 ix 3.13 Two dimensional projection of the intrinsic shapes of the synthetic dsDNAs 59 3.14 Dependence of jM on curvature . 60 3.15 Dependence of jM on length . 61 3.16 The intrinsic shapes of the genomic sequence dsDNAs with varying length 62 4.1 Unlooping kinetics and the shear force approximation . 68 4.2 Loop breakage assay . 73 4.3 Looped state lifetime vs. loop size . 74 4.4 Crankshaft and pivot rotations . 76 4.5 Umbrella sampling with a biased potential . 78 4.6 WLC force vs. LSEC force . 80 4.7 The shear force vs. loop size . 81 4.8 The relationship between linker lifetime and shear force . 82 4.9 The linear regression of the logarithm of loop lifetime vs. shear force . 83 4.10 τ(0) measurement . 85 4.11 The effect of sodium and magnesium salt on strong dsDNA bending . 86 4.12 Kinking probability in DNA minicircles . 90 4.13 J factor comparison . 91 5.1 Single-molecule FRET looping experiment.
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