Effects of salt solutions on DNA micromechanics under tension

EFFECTS OF SALT SOLUTIONS ON DNA MICROMECHANICS UNDER TENSION FU HONGXIA NATIONAL UNIVERSITY OF SINGAPORE 2006 i EFFECTS OF SALT SOLUTIONS ON DNA MICROMECHANICS UNDER TENSION FU HONGXIA (B. Eng. Shandong University of Science and Technology) (M. Eng. Dalian University of Technology) A THESIS SUBMITED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CIVIL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2006 iii ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisors, Professor Koh Chan Ghee and Associate Professor Lim Chwee Teck, who introduced me to a world of research with both intellectually stimulating and engaging. Thanks for their great guidance and encouragement throughout my Ph.D. study. Thanks for the wonderful opportunity for me to work with and learn from them during these years. I would like to thank National University of Singapore (NUS) for the research scholarship and all the research facilities and resources. Especially, I deeply thank the Nano Biomechanics Laboratory (Division of Bioengineering, NUS) for providing great experimental support for my research. I really appreciate the help of the lab officers, Ms. Tan P. S. Eunice, Mr. Hairul Nizam Bin Ramli and Ms. Low Y. H. Kelly. I would like to thank many people who helped me during my Ph.D. study. Chen Hu for the great help to my research. Thank Dr I’m very grateful to him for the discussion and advice on my numerical and experimental studies. Thank Dr Yan Jie for the valuable comments on my thesis. Thank Lee Y.Y., Gregory, Cheong F.C., Li Ang, Qie Lan, Vedula Sri Ram Krishna, Zhou Enhua, Tay C. S. and Ng C. L. for sharing their knowledge and experience for my experiments. Thank Mr. Sit B. C., Mr. Ang B. O., Mr. Yip K. K., Mdm Annie Tan, Mr. Kamsan B. R. and Mr. Wong K. W. for the help during my teaching assistant. Thank Zhao Li, Lee S. C., Chhoa C. Y., Lim K. G., Leong K. S., Chia K. S. and Wang Zengrong for their help and encouragement. I sincerely thank my family for their continuous support and encouragement. ii TABLE OF CONTENTS TITLE PAGE . i ACKNOWLEDGEMENTS . ii TABLE OF CONTENTS iii SUMMARY . vii LIST OF TABLES . ix LIST OF FIGURES x NOMENCLATURE . xiii CHAPTER INTRODUCTION 1.1 DNA Structure . 1.2 DNA Micromechanics . 1.2.1 Introduction of DNA Micromechanics . 1.2.2 Studies of DNA Micromechanics under Tension . 1.3 Objectives and Scope of This Study 13 1.4 Organization of Thesis . 14 CHAPTER LITERATURE REVIEW 16 2.1 Experimental Methods for DNA Manipulation . 16 2.2 Numerical Models for DNA Micromechanics under Tension . 19 2.2.1 Conformational Structures of DNA 20 2.2.2 Numerical Models for Elastic Behavior of DNA . 22 2.2.2.1 FJC Model 22 2.2.2.2 WLC Model . 23 2.2.3 Models for Overstretching Transitions of DNA . 25 2.2.3.1 State Transition Models . 25 2.2.3.2 ZZO Model 27 iii 2.2.4 2.3 Models Dependent on Solution Conditions 30 Summary 33 CHAPTER EXPERIMENTAL SETUP AND PROCEDURES . 35 3.1 Background 35 3.2 Principles of Optical tweezers . 36 3.3 Force Calibration of Optical tweezers . 39 3.3.1 Escape Force Method 40 3.3.2 Trap Stiffness Based Methods 42 3.3.3 Recommended Force Calibration Method 45 3.4 Experimental Setup 47 3.4.1 Single-Beam Optical tweezers 48 3.4.2 Sample Heater . 49 3.4.3 Sample Chamber . 50 3.5 Sample Preparation 50 3.5.1 Preparation of λ-DNA . 50 3.5.2 Binding of DNA & Microspheres . 53 3.6 Experimental Manipulation . 56 3.7 Summary 59 CHAPTER IONIC EFFECTS ON ELASTIC PROPERTIES OF DNA . 60 4.1 Extensible WLC Model Studies 60 4.2 OSF Theory Studies . 63 4.3 Elastic Moduli Renormalization Model Studies 66 4.3.1 Elastic Moduli Renormalization Model 66 4.3.2 Results and Discussion . 69 4.4 Summary 70 iv CHAPTER IONIC EFFECTS ON THE FIRST OVERSTRETCHING TRANSITION OF DNA . 73 5.1 Expeirmental Study of Salt Solution Effects on the First Overstretching Transion 74 5.1.1 Effect of Sodium Ionic Strength . 74 5.1.2 Effect of Magnesium Ionic Strength . 76 5.2 Numerical Study of Salt Solution Effect on the First Overstretching Transiton . 79 5.2.1 Modified ZZO Model . 79 5.2.2 Analytical Results . 84 5.2.3 Metropolis Monte Carlo Simulation . 91 5.2.3.1 Discrete Modified ZZO Model 91 5.2.3.2 MMC Method 93 5.2.3.3 MMC Simulation Results 95 5.3 Summary 95 CHARPTER THE SECOND OVERSTRETCHING TRANSITION OF DNA 100 6.1 Experimental Study of Ionic Effects on the Second Overstretching Transition 100 6.2 Numerical Study of Ionic Effects on the Second Overstretching Transition. 103 6.2.1 Possible DNA Structures during Overstretching Transitions . 103 6.2.2 Three-State Ising-Like Model . 109 6.3 Numerical Study of Other Effects on the Second Overstretching Transition 121 6.3.1 Kinetic Three-State Ising-Like Model 121 6.3.2 Results and Discussion . 124 v 6.4 Summary 129 CHARPTER 7.1 CONCLUSIONS AND RECOMMENDATIONS 131 Summary of Findings and Contributions . 131 7.1.1 Studies on Elastic Properties of DNA . 131 7.1.2 Studies on the First Overstretching Transition of DNA . 132 7.1.3 Studies on the Second Overstretching Transition of DNA . 133 7.2 Future work 134 REFERENCES 136 PUBLICATIONS 149 vi SUMMARY The main objective of this research is to experimentally and numerically investigate the effects of salt conditions on the mechanical properties of single DNA molecules under tension. In particular, the ionic effects of sodium and magnesium salt solutions on the first and second overstretching transitions are examined. Firstly, the elastic properties of DNA are studied by curve fitting of the experimental data with the extensible worm-like chain model, Odijk-Skolnick-Fixman theory and elastic moduli renormalization model. The sodium and magnesium ionic effects on the persistence length, elastic stretch modulus and effective length per charge of DNA are studied. The results show that when the ionic strength of sodium or magnesium salt solution increases, the persistence length and effective length per charge of DNA decreases while the elastic stretch modulus increases. Secondly, a three-dimensional model, namely the modified ZZO model, is proposed to investigate the ionic effects on the first overstretching transition of DNA. In this model, bending deformation of DNA backbones, cooperativity of base-stacking interactions, electrostatic interactions and spatial effects of DNA double helix structure are all taken into account. The electrostatic energy is explicitly given as a function of folding angle and ionic strength. A new parameter is also introduced to account for the cooperativity of base-stacking interactions. The results show that the first overstretching force is linear with the natural logarithm of ionic strength. Finally, using optical tweezers, the ionic effects on the second overstretching transition vii of DNA are experimentally studied. The results show that the second overstretching transition force increases when the ionic strength increases. The second overstretching transition curve is less pronounced for low ionic strengths than that for higher ones. Following Cocco et al. (2004), the three-state Ising-like model is used to study the ionic effects on the second overstretching transition of DNA. In this model, each base pair of DNA is assumed to take one of the three states, which are B-DNA, S-DNA and ssDNA. The proportions of each state during the transition suggest that S-DNA is not one or two unrelated ssDNA but essentially a double-strand DNA with some unpeeled parts and melted base pairs. Furthermore, the kinetic model based on this three-state Ising-like model is applied to study the effects of DNA sequence and stretching speed on the second overstretching transition. The results show that the second overstretching transition force increases when the stretching speed increases. Because the ssDNA free energy or the free energy of stacking interaction of AT-rich DNA is much lower than that of GC-rich DNA, the second overstretching transition is more distinct for GC-rich DNA than that for AT-rich DNA. viii LIST OF TABLES TABLE PAGE Table 3.1 Drag on a particle near chamber surface (Faxen’s Law)*. 41 Table 4.1 Effects of sodium and magnesium ionic strength ( c ) on the persistence length ( A ) and elastic stretch modulus ( S ) of single DNA molecules at 37˚C. 62 Table 4.2 Effects of sodium ionic strength on the persistence length of single DNA molecules (37˚C) calculated by the extensible WLC model and the elastic moduli renormalization model. 72 Table 4.3 Effects of magnesium ionic strength on the persistence length of single DNA molecules (37˚C) calculated by the extensible WLC model and the elastic moduli renormalization model. 72 Table 5.1 Experimental data of the first overstretching forces at different sodium salt solutions and temperatures. 76 Table 5.2 Comparison of the first overstretching transition forces under different sodium and magnesium salt solutions at 37˚C. 78 Table 5.3 Comparison of experimental and analytical results for the first overstretching forces under different sodium salt solutions at 37°C. 90 Table 5.4 Comparison of experimental and analytical results for the first overstretching forces under different magnesium salt solutions at 37°C. 90 Table 5.5 Comparison of experimental and numerical results for the first overstretching forces under different sodium salt solutions at 37°C. 99 Table 6.1 Comparison of persistence length A and stretch modulus S for B-DNA and S-DNA between the data determined by the three-state model and those in Chapter 4. 116 Table 6.2 Stacking free energy of the neighboring base pairs of DNA in 150 mM NaCl with 10 mM Tris and mM EDTA buffer solution at 20°C. 128 ix CHAPTER CONCLUSIONS AND RECOMMENDATIONS 3. 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[2] Fu H.X., Koh C.G., Chen H. and Lim C.T., 2006, Experimental and Numerical Studies on B-DNA Overstretching Transition in Presence of Sodium Ions at Physiological Temperature, Solid State Phenomena, 2006. (Accepted) [3] Lim C.T., Zhou E.H., Li A., Vedula S.R.K. and Fu H.X., Experimental Techniques for Single Cell and Single Molecule Biomechanics, Materials Science & Engineering C: Biomimetic & Supramolecular Systems, 26, pp: 1278-1288, 2006. [4] Chen H., Fu H. X. and Koh C. G., Sequence-dependent Unpeeling Dynamics of Stretched DNA Double Helix, The European Physical Journal E., 2006. (Submitted) Conference Papers [1] Fu H.X., Koh C.G., Chen H. and Lim C.T., Experimental and Numerical Studies on B-DNA Overstretching Transition in Presence of Sodium Ions at Physiological Temperature, China International Conference on Nanoscience and Technology, June 9-11, 2005, Beijing China. [2] Fu H.X., Koh C.G., Chen H., Tay C. S. and Lim C. T., Ionic effects of magnesium 149 PUBLICATIONS salt solution on the elastic&overstretching response of single B-DNA molecules. The 12th International Conference on Biomedical Engineering, December 7-10, 2005, Singapore. (The conference paper [1] has been recommended by the conference committee and accepted to be published in “Solid State Phenomena”.) 150 [...]... 1997) Among all the forms of DNA, B -DNA is thought to be the dominant form of DNA under physiological conditions Many of biological functions of DNA, such as DNA and RNA polymerase interaction, are related to the micromechanics of B -DNA under tension The micromechanics of B -DNA and its extended form S -DNA will be investigated in this research 1.2 DNA Micromechanics 1.2.1 Introduction of DNA Micromechanics. .. modulus of DNA; K (R ) Renormalized bending modulus of DNA; kB Boltzmann constant; L Contour length of DNA; lB Bjerrum length; xiii R Radius of DNA; s Arc length along the backbone of DNA; S Elastic stretch modulus of DNA; S (R ) Renormalized elastic stretch modulus of DNA; ssDNA Single-strand DNA; T Absolute temperature; t Tangential vector of the central axial of DNA; t1 ,t 2 Tangential vectors of the... undoubtedly improve our understanding of the relations of structure, micromechanics and biological functions of DNA The details of modern techniques used to study the mechanical properties of DNA will be introduced in the next chapter In the studies of DNA micromechanics under tension, torsion, unzipping and interaction with proteins, the mechanical properties of DNA under tension are always involved... also dependent on the ionic strength Experimental studies are needed to verify the effects of ionic strengths on the second overstretching transition 12 CHAPTER 1 INTRODUCTION 1.3 Objective and Scope of This Study The main objective of this study is to experimentally and numerically investigate the effects of salt conditions on DNA micromechanics under tension, especially the first and second overstretching... different ionic strengths This study is the first experiment to show the magnesium ionic effects on DNA elastic properties and overstretching transitions under tension The experimental results for the sodium ionic effects on DNA micromechanics can essentially confirm with the previous studies by Wenner et al (2002) 2 The ionic effects of sodium and magnesium salt solutions on the elastic properties of single... the magnesium ionic effects on DNA micromechanics Although Baumann et al give the experimental data for the elastic properties of DNA in 100 µM MgCl2 solution They have not shown how the different magnesium salt concentrations affect the mechanical properties of DNA Therefore, the effects of magnesium salt solutions on the elastic properties and the first overstretching transition of DNA will be investigated... stretching 120 Figure 6.8 Dependence of stretching speed on the force vs extension relationship of homopolymer poly(GC) DNA with N = 500 bps 124 Figure 6.9 Dependence of stretching speed on the force vs extension relationship of homopolymer poly(AT) DNA with N =1000 bps 126 Figure 6.10 Dependence of stretching speed on the force vs extension relationship of a part of λ -DNA from 25,001 bp to 26,000 bp 130...LIST OF FIGURES FIGURE PAGE Figure 1.1 Primary structure of DNA 3 Figure 1.2 Double helix structure of DNA (Karp 1996) 4 Figure 1.3 Schematic diagram of DNA conformation transition under tension 8 Figure 2.1 Scheme diagrams of DNA micromanipulations 18 Figure 2.2 Conformational models of DNA 21 Figure 2.3 Schematic diagram of ZZO model (Zhou et al., 2000a, b) 28 Figure 3.1 Schematic diagrams of optical... transition of DNA This research is expected to enhance our understanding and modeling capability on the effects of salt conditions on DNA behavior in some biological functions, such as DNA wrapping around histones, packing into chromosomes, bending upon interaction with proteins and looping to connect enhancer and promoter regions 1.4 Organization of Thesis In Chapter 2, the literature review of experimental... Chapter 5, the effects of ionic strength on the first overstretching transition of single DNA molecules are experimentally and numerically studied The modified ZZO model is proposed to study the electrostatic contribution of sodium and magnesium cations to the first overstretching transition force In Chapter 6, the ionic effects of NaCl solutions on the second overstretching transition of single DNA molecules . i EFFECTS OF SALT SOLUTIONS ON DNA MICROMECHANICS UNDER TENSION FU HONGXIA NATIONAL UNIVERSITY OF SINGAPORE 2006 ii EFFECTS OF SALT SOLUTIONS ON DNA MICROMECHANICS. the effects of salt conditions on the mechanical properties of single DNA molecules under tension. In particular, the ionic effects of sodium and magnesium salt solutions on the first and second. micromechanics of B -DNA under tension. The micromechanics of B -DNA and its extended form S -DNA will be investigated in this research. 1.2 DNA Micromechanics 1.2.1 Introduction of DNA Micromechanics