Melting dynamics of short dsDNA chains in saline solutions
© He et al. 2015
Received: 30 September 2015
Accepted: 2 December 2015
Published: 15 December 2015
DNA melting has attracted much attention due to its importance in understanding the life-reproduction and metabolism and in the applications of modern DNA-based technologies. While numerous works have been contributed to the determination of melting profiles in diverse environments, the understanding of DNA melting dynamics is still limited. By employing three-site-per-nucleotide (3SPN) double-stranded DNA (dsDNA) model, we here demonstrate the melting dynamics of an isolated short dsDNA under different conditions (different temperatures, ionic concentrations and DNA chain lengths) can be accessed by coarse-grained simulation studies. We particularly show that at dilute ionic concentration the dsDNA, regardless being symmetric or asymmetric, opens at both ends with roughly equal probabilities, while at high ionic concentration the asymmetric dsDNA chain opens at the A-T-rich end. The comparisons of our simulation results to available data are discussed, and overall good agreements have been found.
KeywordsDNA Melting dynamics Molecular simulation Ionic concentration
Denaturation of duplex DNA by heat leading to strand separation is known as DNA melting, and such phenomenon has caused more and more research interests in recent decades due to its importance in understanding the life-reproduction and metabolism. Primarily, a double-stranded DNA (dsDNA) dissociating into two single-stranded DNAs (ssDNA) lays a cornerstone for transcribing genetic information from DNA to RNA (Wang et al. 1998; Rief and Spudich 1999), and this process is indispensable during the replication and transcription of DNA. The DNA denaturation is also utilized in developing modern DNA-based technologies (Mirkin et al. 1996; Jin et al. 2003; Wu et al. 2011; Belozerova and Levicky 2012; Song et al. 2013; Wu et al. 2013). For instances, Jin et al. (2003) showed that the well-defined characteristic of the melting transition enables the detection of single-base mismatches between a probe and target DNAs. By measuring the dissociation rates of dsDNA at different temperatures, Reed and Wittwer proposed a method for tracing food products with High Resolution Melting Analysis (Reed and Wittwer 2004).
DNA melting process is usually characterized with the melting temperature. Melting temperature is conventionally defined as the temperature at which the probabilities of finding a given DNA in the hybridized and dissociated states are the same. The melting temperature is closely related to the environment to which the DNA is subjected (Sorokin et al. 1997; Mrevlishvili et al. 1998; Goobes et al. 2003; Khimji et al. 2013; Nakano et al. 2014). Mrevlishvili et al. (Mrevlishvili et al. 1998) found that the DNA melting temperature increases with the increase of the ionic concentration, and the same conclusion has been drawn by Sorokin et al. through a study on the influences of ions on the thermo-stability of dsDNA (Sorokin et al. 1997). Goobes et al. (2003) added crowding reagent PEG into DNA solution, and found that inertia polymers are in favor with the thermostability of DNA. However, the added reagents may also bring opposite effect due to their molecular interactions with the basepairs in dsDNA. Nakano et al. (2014) found that the melting temperature decreases in the presence of PEG, especially in the region with high PEG concentrations. Different reagents may have different effects on DNA melting depending on the competition between entropic effect and correlation effect. Indeed, Khimji et al. (2013) concluded that the melting temperature of duplex DNA is much higher in polyanions than in non-ionic polymers with similar ionic strength due to an additional electrostatic contribution beyond the excluded volume effect.
Apart from these experimental studies, many coarse-grained theoretical models have been developed to address the DNA melting profile (Drukker and Schatz 2000; Knotts et al. 2007; Sambriski et al. 2009a, b; Liu et al. 2010; Ouldridge et al. 2011; Freeman et al. 2011; De Pablo 2011; Liu et al. 2011, 2012). Drukker and Schatz (2000) proposed a beads-spring model which allows for the investigation of hybridization by using simulation. In this model one nucleotide is represented by two sites, and one is for skeleton and the other for base. The model by Ouldridge et al. (2011) is specifically targeted to reproduce the thermodynamics of DNA melting. However, the model cannot account for electrostatics and sequence specificity. Recently, Knotts and De Pablo et al. (Knotts et al. 2007; Sambriski et al. 2009a, b; Freeman et al. 2011; De Pablo 2011) proposed an advanced three-site-per-nucleotide (3SPN) model and its extensions. This model faithfully captures the characteristics of major and minor grooves and the elasticity of DNA, and it involves an ion-concentration dependent long-range electrostatic interaction between the phosphates in two strands. This model is parameterized by fitting the thermal melting experimental data in a constant ionic strength, and unsurprisingly it can well predict the behaviors of DNA chain including melting profile, bubble formations and the mechanical properties of the molecule as a function of ionic concentration. Besides the simulation models, thermodynamic models are also developed for studying the melting process. Very recently, Liu et al. (2010) developed an interesting molecular thermodynamic model from the perspective of phase equilibrium between the association and dissociation states of each basepair. By constructing a proper free energy function which involves the melting enthalpy contribution from each basepair, they showed that the melting curves and melting temperatures of dsDNA in ionic and crowded solutions or in confined spaces could be successfully predicted (Liu et al. 2010, 2011, 2012).
While numerous experimental and theoretical works have been contributed to the determination of melting profile of dsDNA in diverse environments, the melting dynamics of dsDNA, especially for those with short chain lengths, is less understood (Drukker et al. 2001; Banerjee and Pal 2007; Wong and Pettitt 2008; Kenward and Dorfman 2009). Generally, the access of intermediate melting transition state is of great interest. Such information is crucial because it’s helpful to explore the sequence and end effects and to reveal the role of crowding or confinement environment for DNA melting (Perez and Orozco 2010; Toma et al. 2014).
The aim of present work is to study the dynamics of DNA melting in dilute brine environment. The denaturation of short DNA chains is usually employed in Biosensing (Sendroiu et al. 2011; Toma et al. 2014; Loget and Corn 2014) and grafting or anchor technology (Ma et al. 2009; Seela et al. 2011; Zhao et al. 2012; Woller et al. 2012). Besides, it is straight-forward to explore the mechanism of DNA melting by using short DNA chains than using long DNA chains (Wong and Pettitt 2008; Miyoshi and Sugimoto 2008).
Modelling and simulation details
The geometry and force field of 3SPN model are detailed elsewhere (Knotts et al. 2007; Sambriski et al. 2009a, b), and here we only give a brief introduction. As shown in Fig. 1a, the 3SPN dsDNA model is composed of two ssDNA strands, and in each strand three kinds of beads are included which represent sugar, phosphate and nucleotide. Each phosphate bead carries a negative elementary charge, and the nucleotide can be Adenine (A), Guanine (G), Cytosine (C) or Thymine (T). For different basepairs, i.e., A-T and G-C, the hydrogen bonding interactions are distinct. At natural state, the geometry of dsDNA can be characterized by the lengths of the connecting bonds and the values of the triangle and dihedral angles. Besides, the backbones of the second chain should be reproduced by simply rotating the first chain along the axis and then shifting up with a given height for a double helix structure. Under perfect matching condition, the structure of a dsDNA is determined once the sequence of nucleotides in either strand is specified, and in this circumstance we conventionally use a sequence composed of A, G, C and T to represent a dsDNA chain.
The interactions involved in 3SPN model include three sets of contributions. The first one is intra-strand interaction accounting for the structural stability of single strand, and the second one accounts for the interaction between two strands involving the electrostatic repulsion between the phosphates in both strands and the basepairing interaction. This baseparing interaction is responsible for the hybridization, and provides a key energy barrier from DNA melting. The last contribution accounts for the elasticity of DNA chain, and specifically the electrostatic interaction among the phosphate beads are included, and the strength of this solvent-mediate interaction is dependent on the ion-concentration in solution. The mathematical formats of those interactions and the involved parameters can be found in the original work (Knotts et al. 2007; Sambriski et al. 2009a, b).
Sequences of DNA chains considered in this work
Sequence (5′ to 3′)
ATC CGT ATG CG
ATC CGT ATG CGA TCC G
ATC CGT ATG CGA TCC GTA TGC
ATC CGT ATG CGA TCC GTA TGC GAT CC
The reasons of choosing above target DNA samples are as follows: firstly, DNA chains of different chain lengths should be considered in order to access the length effect on DNA melting dynamics. Secondly, the sequence effect should be reflected, and thus the other three dsDNA samples are generated by simply repeating, in a whole or half manner, the sequence of the first sample. For simplicity, the four DNA chains are referred to as 11, 16, 21 and 26 bp. The melting temperatures of these dsDNA samples are reported to be within the range of 290–350 K depending on the chain length and the salt concentration (Owczarzy et al. 2004; Freeman et al. 2011).
To implement the force calculation during simulation, we introduce the following method to label the beads in dsDNA chain (see Fig. 1a for illustration): the beads in the first strand are labeled by odd numbers, and the beads in the second strand are labeled by even numbers. In each strand, the beads are labeled from the bottom up and from nucleotide to sugar site.
The initial configuration of the DNA is generated following the method described by Knotts et al. (2007). The differential equation above is solved by using Velocity-verlet method iteratively with a time step of 0.01 ps. The simulation is started with 40,000 steps (0.4 ns) of steepest descent minimization followed by a 1 ns equilibration process during which the system temperature is set as room temperature. After equilibrium, a temperature jump to the final target temperature is carried out (Qamhieh et al. 2009) for studying the melting dynamics. The target temperatures, though high, are chosen to ensure the occurrence of melting within microsecond timescale. Under a given condition, 100 parallel simulations are run, and then the melting time is averaged and analyzed. Similar to the original work (Knotts et al. 2007), when the distance of basepair G-C is larger than 2.8694 Å or that of basepair A-T is larger than 2.9002 Å, and in addition two nucleotide beads move in opposite direction, the basepair is considered as being dissociated. Three factors that may affect the DNA melting dynamics are investigated including temperature, ionic concentration and chain length.
Results and discussions
Effect of temperature
The comparison in Fig. 3 shows that a qualitatively agreement between our simulation and Qamhieh’s result can be found, i.e., the speed of basepair dissociation generally becomes larger and larger during the melting process. One may notice that the time scale of melting duration time in our study is much smaller than that in Qamhieh’s work, and this is very likely due to the difference in modelling, i.e., in Qamhieh’s work the dsDNA chain was described by using an atomistic model while in current work the DNA dissociation process is studied by using a coarse-grained model. Although coarse-grained simulation can give qualitatively or semi-quantitatively similar results as the corresponding all-atom simulation, the simulation time scales in both systems are generally believed to be different. In addition the dsDNA being tethered may present different melting dynamics as in a free space because the tethered dsDNA encounters an extra entropic force from the presence of wall (Zhao et al. 2011). Similar agreement can be obtained by comparing our simulation results to those in Perez’s work. Perez and Orozco (2010) performed a simulation study to gain the real-time atomistic description of DNA unzipping, and they investigated the time evolution of the total number of hydrogen bonds for short dsDNA chains (12-mer) at 368 K, which can be easily converted to the time evolution of total number of separated basepairs. This comparison is very similar to Fig. 3 and thus is omitted here. The agreement with the atomistic simulation results rationalizes our calculation and the analysis on the melting dynamics below.
Effect of sodium ion concentration
κD and Vqq in different ionic concentrations (360 K, 11 bp)
Ionic concentration/mol L−1
Vqq/× 1016 J
Effect of chain length
In the present work, a series of coarse-grained molecular dynamics simulations have been performed for investigating the melting dynamics of short dsDNA chains. By accessing the temperature effect, ionic concentration effect, and chain length and sequence effects, we find that the melting dynamics of short dsDNA chain is no trivial. The addition of ion can improve the stability of DNA. Moreover, the DNA melting has two different pathways: opens at one end, and opens at two ends. At dilute ionic concentration, the dsDNA, regardless of being symmetric or asymmetric, opens at both ends with roughly equal probabilities; while at high ionic concentration, the asymmetric dsDNA chain opens at the A-T-rich end. The comparisons of our simulation results with available data are discussed, and overall good agreements have been found.
The current work reveals the melting dynamics of an isolated short dsDNA chain in saline solution. The majority of DNA in experimental study is long DNA chains and the physiological environment is crowded and geometrically confined. The melting dynamics in practical may be significantly distinct due to the additional steric effect and correlations between DNA chains. Toward that realistic study, a more complex modelling and simulation is required which represents the further direction of this work.
SZ and HL conceived this study. YH carried calculation, and all authors performed data analysis. SZ and YH wrote the manuscript. All authors read and approved the final manuscript.
This work is supported by the National Key Basic Research Program of China (2014CB748500), National Natural Science Foundation of China (Nos. 21173079, 21206036), and the 111 Project of China (No. B08021) and the Fundamental Research Funds for the Central Universities of China. SZ also acknowledges the support of the Shanghai Science and Technology Committee Rising-Star Program (No. 14QA1401300).
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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