Microarray based real-time analysis of nucleic " acid hybridization kinetics and thermodynamics\ Dissertation Zur Erlangung des Grades Doktor der Naturwissenschaften Am Fachbereich Biologie Der Johannes Gutenberg-Universit¨atMainz DI Siegfried Krainer geb. am 21.01.1968 in Friesach/Osterreich¨ Mainz, 2011 Contents Contents i List of Figures v List of Tables vii 1 Introduction 7 1.1 Motivation ....................................... 7 1.2 The topic ........................................ 7 1.3 Organization of the content.............................. 8 2 Physical chemistry of nucleic acid hybridization9 2.1 Introduction....................................... 9 2.2 Building blocks of DNA and RNA .......................... 9 2.2.1 D-Ribose: Determining structure and chirality ............... 10 2.2.2 Sugar pucker and nucleic acid structure ................... 11 2.2.3 Locked nucleic acid (LNA) .......................... 12 2.2.4 Bases: The letters of the genetic code .................... 14 2.2.5 Interaction between bases........................... 15 2.2.6 The phosphate backbone ........................... 18 2.2.7 Structural parameters of nucleic acids .................... 18 2.2.8 Base opening in RNA and DNA ....................... 18 2.2.9 Influence of the C5-methyl group....................... 21 2.3 Charge distribution of bases.............................. 24 2.3.1 Protonation and ionization of Nucleic Acid bases.............. 25 2.3.2 Calculation of the ground state charge distribution............. 25 2.3.3 The interaction with ionic solution...................... 25 2.4 Physical characterization of nucleic acids....................... 28 2.4.1 Physics of biopolymers............................. 28 2.4.2 Parameters characterizing biopolymers.................... 28 2.5 Physical chemistry of hybridization.......................... 30 2.5.1 Immobilization chemistry on aldehyde surfaces ............... 30 2.5.2 Immobilization of DNA on bare glass .................... 31 2.6 Physics of online hybridization measurement .................... 32 2.6.1 Probe density on microarrays......................... 33 2.6.2 Target and dye concentration......................... 33 i ii CONTENTS 2.6.3 Hybridization efficiency ............................ 34 2.6.4 Absorption of the incoming photons ..................... 35 2.6.5 Emission intensity of the fluorophores .................... 35 2.6.6 Influence of photobleaching.......................... 36 2.6.7 Influence of the optics............................. 36 2.6.8 Confocal online measurement......................... 36 2.6.9 Efficiency of an EM-CCD camera....................... 37 2.6.10 Quantitiative description of online measurment............... 37 2.7 Summary ........................................ 38 3 Thermodynamics of hybridization 41 3.1 Introduction....................................... 41 3.2 Parameters influencing melting temperature..................... 42 3.2.1 Oligonucleotide length and sequence..................... 42 3.2.2 Salt concentration ............................... 42 3.2.3 Oligonucleotide concentration......................... 42 3.2.4 Denaturating Agents.............................. 42 3.2.5 Influence of metal ions on nucleic acid hybridization............ 43 3.2.6 Simple formulas for Tm ............................ 44 3.3 Thermodynamic of Hybridization........................... 46 3.3.1 Theory of the two-state model ........................ 46 3.3.2 Temperature dependence of thermodynamic properties........... 47 3.3.3 Solution based results ............................. 49 3.4 The Nearest Neighbor model ............................. 49 3.4.1 Parameters of the NN model ......................... 49 3.4.2 Gibbs free energy................................ 50 3.4.3 Comparison of NN results with simple formulas............... 51 3.4.4 Extraction of NN parameters from experimental data ........... 54 3.4.5 The influence of mismatches.......................... 54 3.4.6 Influence of probe length on melting analysis ................ 55 3.5 Thermodynamic changes during melting....................... 55 3.5.1 Introduction .................................. 55 3.5.2 Calorimetric parameters of DNA melting .................. 59 3.6 Thermodynamics of DNARNA hybrids....................... 59 3.6.1 Structure and thermodynamics of DNARNA duplexes .......... 60 3.7 Thermodynamic of surface adsorption ........................ 61 3.7.1 The Langmuir theory of adsorption...................... 61 3.7.2 Is Microarray hybridization a Langmuir process?.............. 61 3.8 Publication: Solid phase high resolution melting .................. 62 3.8.1 Abstract..................................... 62 3.8.2 Introduction .................................. 62 3.8.3 Materials and Methods ............................ 63 3.8.4 Results and discussion............................. 65 3.8.5 Conclusion ................................... 68 3.8.6 Acknowledgement ............................... 69 4 Kinetic of Nucleic Acid Hybridization 71 4.1 Diffusion of nucleic acid molecules .......................... 71 4.1.1 Influence of DNA length on mobility..................... 71 CONTENTS iii 4.1.2 Estimation of the diffusion coefficient of DNA................ 72 4.1.3 Diffusion coefficient in solution and in cytoplasm.............. 73 4.1.4 Temperature behavior of diffusion ...................... 74 4.2 Physics of diffusion-reaction systems......................... 74 4.2.1 Is DNA hybridization diffusion or reaction controlled?........... 74 4.3 Solution based results for hybridization ....................... 75 4.4 Numerical solution of diffusion-reaction equation .................. 77 4.4.1 Introduction .................................. 77 4.4.2 The diffusion equation............................. 77 4.4.3 Finite-difference algorithm for diffusion equation.............. 78 4.4.4 Crank-Nicolson algorithm in one dimension................. 79 4.4.5 Crank-Nicolson algorithm in two dimension................. 79 4.4.6 Crank-Nicolson in cylinder coordinates.................... 79 4.4.7 Modeling of denaturation ........................... 81 4.5 Conclusion ....................................... 82 5 Algorithms and modeling tools 83 5.1 DNA hybridization as a Fermi-Dirac system..................... 83 5.1.1 Introduction .................................. 83 5.1.2 Fermi-Dirac algorithm............................. 84 5.1.3 Experimental data............................... 84 5.2 Modeling with ChipCheckII.............................. 85 5.3 Publication: Physical model based algorithm .................... 87 5.3.1 Abstract..................................... 87 5.3.2 Background................................... 89 5.3.3 Methods..................................... 90 5.3.4 Results and discussion............................. 92 5.3.5 Kinetic analyis................................. 92 5.3.6 Conclusion ................................... 95 5.3.7 Competing interests .............................. 96 5.3.8 Author's contribution ............................. 96 6 Competitive hybridization of DNA and RNA 103 6.1 Introduction.......................................103 6.2 The influence of target concentration.........................103 6.3 Kinetics of DNADNA competitive Hybridization..................104 6.4 Kinetics of DNARNA competitive Hybridization..................105 6.4.1 Hybridization of RNA to double-stranded DNA . 105 6.4.2 Strand displacement kinetics .........................106 6.5 Publication: Competitive Hybridization .......................109 6.5.1 Abstract.....................................109 6.5.2 Introduction ..................................110 6.5.3 Methods.....................................111 6.5.4 Theoretical models...............................112 6.5.5 Results and discussion.............................114 6.5.6 Conclusion ...................................118 7 Discussion 123 7.1 Physics of real-time measurement...........................123 iv CONTENTS 7.2 Hybridization isotherms................................123 7.3 High resolution melting analysis............................123 7.4 Competitive hybridization...............................124 7.5 Influence of the C5-methyl group...........................124 7.6 Outlook.........................................125 Bibliography 127 A Probe and target sequences 143 A.1 Phylogenetic tree of actin genes............................143 A.2 Sequences of the Actin X-chip.............................143 A.2.1 Sequences of the Actin genes .........................143 A.2.2 Sequences of the PCR primers ........................146 A.2.3 Sequences of the oligo probes.........................146 A.3 Similarity matrix of the Actin genes .........................150 List of Figures 2.1 Structure of an RNADNA hybrid............................ 10 2.2 D-ribose and the enantiomer L-ribose........................... 11 2.3 Formation of β-ribose ................................... 11 2.4 Schematic representation of DNA phosphodiesterbonds................. 12 2.5 C20-endo and C30-endo conformation........................... 13 2.6 Stabilization of C30-endo puckering in RNA....................... 13 2.7 Structure of LNA in a DNALNA ............................ 14 2.8 α − D−LNA vs β − L−LNA ............................... 15 2.9 The elementary particles of life.............................. 16 2.10 Watson-Crick basepairing and stacking.......................... 16 2.11 Imino and enol form of an AT-basepair ......................... 17 2.12 Stacking of two guanine bases..............................