UNIVERSITA DEGLI STUDI DI PADOVA

Sede Amministrativa: Universit` degli Studi di Padova a Dipartimento di Fisica Galileo Galilei D OTTORATO DI R ICERCA IN F ISICA C ICLO XVI

STATICS AND DYNAMICS OF DNA DENATURATION IN PRESENCE OF A BOUNDARY

Coordinatore: Ch.mo Prof. Attilio Stella Supervisore: Ch.mo Prof. Attilio Stella Co-tutore: Dr. Enzo Orlandini

Dottorando: Giorgio Lissandron

31 Ottobre 2003

Ciao

A mio pap` a

Contents
Introduction 1 Biophysics and DNA 1.1 DNA structure . . . . . . . . . 1.2 Biological Function of DNA . 1.3 DNA-Microarray . . . . . . . 1.4 Single Molecule Manipulation 3 9 9 10 12 14 17 17 17 20 24 27 27 28 31 34 37 39 39 40 41 44 45 47

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2 Statistical models of DNA 2.1 Models a la Poland and Sheraga . ` 2.1.1 Poland and Sheraga model 2.1.2 Polymer networks picture 2.2 Other DNA models . . . . . . . . 3 Adsorption of DNA on a surface 3.1 Introduction . . . . . . . . . . . 3.2 Model and Phase diagram . . . . 3.3 Numerical analysis . . . . . . . 3.4 Polymer Network on the surface 3.5 Discussion . . . . . . . . . . . . 4 DNA hybridization on the surface 4.1 Introduction . . . . . . . . . . . 4.2 The model . . . . . . . . . . . . 4.2.1 Loop length distribution 4.3 End-segment distribution . . . . 4.3.1 Network prediction . . . 4.4 Entropic Force . . . . . . . . .

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5 Kinetics of DNA denaturation 53 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 5.2 Model and numerical results . . . . . . . . . . . . . . . . . . . . 54 5.2.1 Kinetics of DNA denaturation . . . . . . . . . . . . . . . 54 1

2 5.2.2 Conclusion A Lattice models for polymer physics B Monte Carlo methods for polymers

CONTENTS Kinetics of DNA hybridization . . . . . . . . . . . . . . . 59 63 65 69

Introduction
In the past few years there has been an explosion of biological data, such as the complete genome of many organisms, the sequence of DNA macromolecules and numerous proteins, and the expression proles of thousands of genes by chip technology. Converting this enormous amount of data to useful biological knowledge requires a multitude of computational and statistical tools, as well as novel conceptual perspectives. Progress in this task also requires addressing issues in optimization, pattern recognition, collective behaviors, which are in the domain of statistical physics. Indeed, a central goal of statistical physics is to describe how complex behavior emerges from the interaction of large numbers of basic elements. Statistical physicists have a long tradition in the study of complex systems. They have developed theoretical concepts and analytical methods that have proved to be extremely powerful in the comprehension of phase transitions, disordered system etc ... The study of the conformational properties of polymers plays a central role both in the development of the statistical mechanics of complex systems and in its potential applications to biophysics at molecular level. Polymers are macromolecules composed by a large number of monomers arranged either in linear chains, or in more complex branched structures. Important advances in the study of their equilibrium and dynamical properties have been obtained using coarse grained descriptions. Indeed, at length scales much larger than that of the monomer units, many properties of polymers result largely independent of the local chemical details. In a most schematic way, a linear polymer in good solvent is often modeled by a self-avoiding walk, (SAW),on a lattice [73]. This is a random walk, which takes into account the effect of excluded volume between the monomers [App. A]. One of the most important theoretical contributions of statistical mechanics to the eld of polymers is an elegant correspondence between a and a magnetic system. In the late sixties, de Gennes performed the relevant mapping of the SAW onto the formal limit of the spin model. Thus, this two different systems, apparently unrelated to each other, are formally governed by the same universal scaling laws in the limit of large spatial scales and long chains, and so fall in the same universality class, quantitatively specied by suitable critical exponents. The advantages from the introduction of simple mod3

els are various: while preserving the physically relevant informations, they allow many steps forward in the theory and in the computer simulations. This approach has been very useful to study homopolymer chains (i.e. polymers for which the basic constituents are monomers of the same kind). In particular in the last fteen years there have been important advances in the comprehension of the transition from the high temperature swollen phase to the low temperature one, where the polymer assumes compact congurations. This transition is known as -collapse and has been extensively investigated by theoretical and numerical methods [71]. Another problem that attracted the attention of the polymer physicists has been the adsorption of a homopolymer on a boundary. Simplied models for the study of polymers near a surface have been studied extensively in last decades by Monte Carlo simulations, mean eld approaches, eld theoretical calculations [71]. So, the phenomenon of homopolymer adsorption, on an attracting wall, is by now well understood. In recent years the interest on polymer statistics focused on heteropolymers. These are systems composed by various kinds of monomers interacting differently among themselves and with the solvent. The study of the conformational properties of those systems poses new numerical and theoretical challenges compared to standard homopolymers [2]. Examples of heteropolymers are proteins, DNA, RNA i.e. the basic constituents of biological matter in living organisms. In this thesis we focus our attention to the conformational properties of the DNA double stranded macromolecule [Cap. 1]. An important conformational transition of DNA is the unbinding process that occurs when the DNA double helix opens up and splits into two separate strands. A DNA molecule may pass from a double-stranded to a single stranded state, either under the effect of an increase in temperature (thermal denaturation1) or through an applied force at one end of the chain (mechanical unzipping). In the two processes that we mentioned we are still in front of an energy-entropy competition: in the rst case the DNA holds together at low temperatures, but when the temperature raises the entropy takes over and the strands unbind. In the second example at xed temperature an external force enters in the energy-entropy competition by favoring energetically congurations that in the previous case were favored only by entropy. The experiments monitor easily the denaturation process because the breaking of the base pairs is accompanied of a large increase of the adsorbance UV light. Plots of the adsorbance as a function of temperature show a multistep behavior consisting of plateaus of variable sizes separated by jumps. This behavior has been interpreted as a signal of rst order transition characterized by a sharp opening of base pairs. In the characterization of such transition and in the determination of its universal features, substantial progresses have been made recently by application of models of polymer physics. [22, 39, 65].
1

The opposite process is called hybridization

The problem of DNA denaturation [see Cap. 2] attracts the attention of statistical physicists since the beginning of the sixties. In a seminal work, Poland and Sheraga ( ), proposed an approximate representation of the DNA molecule undergoing denaturation as a sequence of alternating bound segments and denaturated loops represented by closed simple random walks. They found that the denaturated loop length is the relevant random variable for the characterization of the thermodynamic behavior of a long DNA molecule in solution. Indeed, the distribution of loop lengths follows a power law behavior, , and the exponent determines the nature of the transition. A detailed analysis reveals that the transition is a second order if and rst order if . The model, in which the loops are treated as independent closed random walk, displays a continuous phase transition both in two and three dimension. This approximate model is not adequate to explain the experimentally observed rst order character of the transition. Recently Kafri, Mukamel and Peliti [22] proposed a new approximation in the framework of PS model, that takes into account the effect of self-avoidance between the loops and the rest of the chain. Their approach makes elegant use of theoretical results for the entropy of polymer networks obtained by Duplantier [24] and leads to expect that, indeed, excluded volume constraints are responsible for the discontinuous character of denaturation transition. They model a DNA molecule undergoing a denaturation transition by a suitable polymer network conguration. With this approximation, they obtain , consistent with the rst order character of the denaturation transition. This predictions has been conrmed by a direct determination of based of Monte Carlo methods for a model of DNA on cubic lattice. Most of the statistical mechanical studies of DNA performed so far consider the macromolecule in the bulk situation. Nevertheless, in biophysical experiments and in biological applications the DNA molecules are usually attached on a substrate. For example, in DNA-microarray tecnology [9] single stranded (ssDNA) chains of identical sequence, are anchored to a support interface, then the chains hybridize with free ssDNA in solution, with a complementary sequence. In this case, the surface acts ideally as an impenetrable boundary. In other experiments the DNA molecules turns out to be adsorbed on a substrate [44]. In this latter case, the interactions with the surface could be crucial in determining the conformational properties of the DNA. Its interesting to study the possibility that a competition between the attractive interaction of the substrate and the base pair binding interactions could inuence the denaturation transition. A general aim of this thesis is to introduce and study relatively simple models which describe DNA denaturation in presence of a boundary surface. We investigate by Monte Carlo methods (see App. B) or, if possible, by analytical conjectures, the conformational properties of these models of DNA macromolecules. A rst problem we address is the DNA denaturation in presence of an attractive surface. We introduce a simplied model in which the DNA hybridization occurs 5

only if the molecule is adsorbed on the surface [see Cap. 3] on the other hand, in our model the adsorption transition takes place simultaneously with the denaturation transition. This is far from exhausting all the relevant physical aspects one should take into account in describing the DNA adsorbed on a substrate. One should rather regard our study as a rst step in the statistical mechanics of this problem. We restrict ourselves to describe in the most schematic terms the results of a competition beet ween adsorption and denaturation as far as the conformational properties in the long chain limit are concerned. Our numerical simulations show that the presence of an attracting surface has the effect of sharpening the denaturation transition with respect to the bulk situation. In order to explain this effect we propose a polymer network representation for the system undergoing the denaturation transition which takes into account the presence of the boundary. In particular, we use results of the theory of homopolymer networks attached on a surface, introduced by Duplantier [45, 25], to predict the algebraic decay of the distribution of the loops localized near the surface. Therefore, in our model the presence of a selective interaction between the DNA macromolecule and the substrate seems to modify strongly the conformational properties of DNA. Another issue which we address in this thesis, is the possibility that a mismatch (i.e. a portion of the chain when the bases not have the theirs complementary) in the sequences of the single strand segment could inuence the denaturation properties of the DNA molecules grafted on a surface [ Cap. 4]. The study of this phenomenon results important for the comprehension of the physical behavior of DNA-microarrays and in general for the behavior of the biomolecules attached on a surface. Experiments [41] show that the hybridization process is sensitive to the presence of base pair mismatches along the DNA chain as well as to the position at which those mismatches occur along the grafted strand. In order to simplify this problem, in our model, the substrate acts as a boundary, i.e. we dont consider any specic interaction between the substrate and the molecule. The second approximation concerns the type of mismatch that we consider. The one we treat is due only to the different lengths of the two strands and the excess in length of one of the two, which correspond to the mismatch, is localized at its end, not in the middle, like in some experimental situations [41]. At the denaturation transition, we expect that the resulting relevant topology for the DNA is the -fork near one of the ends of the chain, with two unzipped end-segments. In particular, we are interested to study if the presence of the surface and of the mismatch can modify the probability distribution of the end-segments with respect to the bulk case and to the fully matched case. We analyze numerically the distribution of end-segment length, , and we extend the description to this situation. Our results indicate that the simultaneous presence of the surface and of the chain mismatched promotes the end opening of the macromolecule this effect seems analogous at a repulsive force localized near the surface. The last study in this thesis [see Cap. 5] concerns the numerical investiga6

tion of the kinetics of the DNA denaturation and hybridization processes, for a macromolecule attached on a surface. The kinetics of these processes have attracted much attention in the last few years in view of its connections with single molecule manipulations (i.e mechanical unzipping experiments) and with the DNA microarray technology. Recently some theoretical studies of dynamics of DNA denaturation have been carried out using directed polymer models in the continuum [65, 39]. Further, very recently Marenduzzo et al. [64], studied the denaturation dynamics of a set of exactly solvable lattice models in which the DNA strands are described by two directed walks. The last authors observed peculiar scaling behaviors in different regions of the phase diagram. The exponents of these scalings obey relations which are analogous to those obeyed by dynamical surface growth models. In our work we study both the kinetics of the denaturation (unzipping) and the kinetics of hybridization process for a model which, unlike those quoted above, fully takes into account the excluded volume effects and the presence of an impenetrable boundary. Our numerical results seem to indicate that the two processes obey a form of dynamical which is not as simple as in surface growth models. Thesis organization Chapter 1 : We review the main biological characteristics of DNA molecule and the main conformational transitions of the DNA which are relevant for our study. Chapter 2: We describe the Poland and Sheraga model for DNA denaturation transition and it extensions. Chapter 3 : The model for a DNA attached on an attractive boundary is described. In particular we focus our attention to a network picture that characterize the denaturation transition. Chapter 4: Here we study the DNA denaturation in presence of a mismatched along the chain. Chapter 5: We study numerically the scaling properties of the kinetics of DNA denaturation and hybridization App. A: We briey review some properties of lattice model of polymers. App. B: This appendix contains a review of the numerical technique using in our simulations.

WEB bibliography: There are many useful resources on the web, the following links provide background and informations on biophysics: 7

CONTENTS Kavli Institute of Theoretical Physics, Program on Bioinformatics (2001): http://matisse.ucsd.edu/itp-bioinfo/ A course on Statistical Physics of Biological Information and Complexity http://guava.physics.uiuc.edu/ nigel/courses/498BIO/ A course at Berkeley on Molecular Biophysics http://alice.berkley.edu The Nordita course of Physics of Molecular Biology: http://www.nordita.dk/ sneppen/biocourse/problems.html