MELTING TEMPERATURE AND STABILITY CALCULATIONS OF

AN OLIGONUCLEOTIDE IN SOLUTION AND ON SURFACE

USING AN INTEGRATED THERMODYNAMICAL APPROACH

Melting temperature is defined as the temperature at which 50% of all the duplexes are denatured. It is an important factor in many applications used in molecular biology, such as, PCR, hybridization, northern and southern blots, and sequencing. Its significance comes from the fact that all of these applications utilize hybridization or annealing temperature 5- 10ºC lower than the melting temperature for optimum performance.

With the need to find the melting temperature of an oligonucleotide, various experimental and theoretical methods are introduced to the service of the scientific community. Experimental methods are based on the production and analysis of annealing/ melting curves of the oligonucleotide under investigation. These curves aid in screening helix to coil or coil to helix transitions of the oligonucleotide by plotting the signal measurements of the denaturing or annealing reaction with respect to temperature at a constant wavelength, i.e. 260 nm. Signals can vary from UV absorbance to fluorescence emission, or to the intensity of an NMR peak or Raman signal.

Limiting the analysis of a melting/ annealing profile to a simple melting temperature plot examination is too restrictive. It is well known that strength of a folded conformation determines the stability of the duplex. So, at this point, the thermodynamical properties, i.e. ΔHº (Standard Enthalpy of helix formation at a given temperature), ΔSº (Standard Entropy of transition at a given temperature) and ΔGº (Standard Gibbs Free Energy of helix formation at a given temperature) come into play. Graphical methods based on reciprocal melting temperature vs concentration plots lead to the well-known semi-empirical melting temperature calculation, Nearest Neighbor Method.

Despite having an experimental basis, Nearest Neighbor Method is preceded by different theoretical melting temperature calculations of short oligonucleotide sequences; Wallace Rule (Marmur and Doty 1962), which accounts for the relative content of the nucleotide sequence; and Salt-Corrected Equation (Wetmur 1991), which brings an improvement by adding a correction factor for the salt concentration. In 1974, it was suggested that not only the relative amounts of the nucleotides; but also the sequential arrangement of different nucleotides plays a major role in the melting temperature (Borer, Dengler et al. 1974). The so-called Nearest-Neighbor Method postulates that the free energy for duplex formation depends on mainly three factors: the initiation free energy given by the unfavorable entropy due to the loss of translational freedom, and the sum of the complementary pair-wise terms between the oligonucleotide sequences as the propagation continues, and the entropic penalty of the maintenance of the symmetry if a self-complementary sequence is investigated. To date, several different experimental thermodynamic parameters for the 10 ‘nearest-neighbor’ di-nucleotide sequences, namely dAA/dTT, dAT/ dTA, dTA/dAT, dCA/dGT, dGT/dCA, dCT/dGA, dGA/dCT, dCG/dGC, dGC/dCG, dGG/dCC have been published (Gotoh and Tagashira 1981; Vologodskii, Amirikyan et al. 1984; Breslauer, Frank et al. 1986; Delcourt and Blake 1991; Doktycz, Goldstein et al. 1992; SantaLucia, Allawi et al. 1996; Allawi and SantaLucia 1997). Since the sequence of the nucleotide is being considered as important as its composition, other conditions need to be taken care of in the melting temperature calculation, including but not restricted to having mismatches in different locations along the sequence (Allawi and SantaLucia 1997; Allawi and SantaLucia 1998; Allawi and SantaLucia 1998); the nature of the ends of the sequence (Bommarito, Peyret et al. 2000). Improvements are constantly being made on the Nearest-Neighbor Method, one being the proposition and derivation of a single and unified set of thermodynamical doublet parameters (SantaLucia 1998). A recent study compares the melting temperature values calculated by the different nearest neighbor thermodynamic parameters in a large set of randomly generated oligonucleotides (16-30mers, 10 different GC contents) to highlight the magnitude of the existing differences and variations that can surface due to the experimental biases introduced while calculating the doublet thermodynamical properties (Panjkovich and Melo 2005).

An alternative approach to predict the melting temperature of a duplex is to start from the calculated or observed molecular properties of the nucleotides, such as their thermodynamical as well as the molecular crystal structure data. My approach takes into account the bond interactions inter- and intra-strand by using a conformational stability value (the difference between the free energies of the single and double stranded states), which separates the free energy into various component energy terms, namely, hydrophobic, base stacking, hydrogen bonding, van der Waals, electrostatic and a tri-nucleotide-level helix stiffness. The duplex initiation, duplex rigidity, symmetry correction and end effects are all accounted for in the model, and multiple linear regression equations are already set up to predict the transition free energy using the individual energy components (Sundaralingam and Ponnuswamy 2004). Calculation of the Gibbs free energy is followed by the utilization of the Gibbs-Helmholtz Equation into the play, which requires the calculation of standard enthalpy and entropy changes. The melting temperature can, then, be predicted using the van’t Hoff Equation. However, as the latest studies show the dependency of the heat capacity term on the temperature, salt concentration, base-pair composition and sequence (Chalikian, Volker et al. 1999; Rouzina and Bloomfield 1999; Rouzina and Bloomfield 1999; Wu, Nakano et al. 2002), further modifications are included, and concurrently screened for accurate estimation of the stability, and thus the melting temperature.

The improvements on the melting temperature prediction are not limited to the semi-empirical approaches mentioned previously. With the launch of the microarrays, various question marks have started to come up in different aspects of the technology. One is the correct prediction of the melting temperature of the oligonucleotides on the surface. All of the methods used rely on the application of the solution thermodynamics, and thus approximating the real melting temperature of the duplex formed on the surface during hybridization. The second part of my research focuses on the experimentally-based theoretical prediction of the melting temperature of the duplexes forming on the surface. To investigate this problem, an array platform, previously developed with the collaboration of our colleagues (LeProust, Pellois et al. 2000; Gao, LeProust et al. 2001; Gao, Gulari et al. 2004), will be utilized to screen the thermodynamical properties, and predict the related parameters of the oligonucleotide duplexes forming on the surface. This procedure includes many steps ranging from the design of the probes on the surface to the analysis of the microarray data obtained after the process is completed.

With this research, a new approach to predict the melting temperature and the stability of the oligonucleotides both in solution and on surface is to be developed using an integrated thermodynamical approach verified by experimental results.

References:

Allawi, H. T. and J. SantaLucia, Jr. (1997). "Thermodynamics and NMR of internal G.T mismatches in DNA." Biochemistry 36(34): 10581-94.

Allawi, H. T. and J. SantaLucia, Jr. (1998). "Nearest neighbor thermodynamic parameters for internal G.A mismatches in DNA." Biochemistry 37(8): 2170-9.

Allawi, H. T. and J. SantaLucia, Jr. (1998). "Thermodynamics of internal C.T mismatches in DNA." Nucleic Acids Res 26(11): 2694-701.

Bommarito, S., N. Peyret, et al. (2000). "Thermodynamic parameters for DNA sequences with dangling ends." Nucleic Acids Res 28(9): 1929-34.

Borer, P. N., B. Dengler, et al. (1974). "Stability of ribonucleic acid double-stranded helices." J Mol Biol 86(4): 843-53.

Breslauer, K. J., R. Frank, et al. (1986). "Predicting DNA duplex stability from the base sequence." Proc Natl Acad Sci U S A 83(11): 3746-50.

Chalikian, T. V., J. Volker, et al. (1999). "A more unified picture for the thermodynamics of nucleic acid duplex melting: a characterization by calorimetric and volumetric techniques." Proc Natl Acad Sci U S A 96(14): 7853-8.

Delcourt, S. G. and R. D. Blake (1991). "Stacking energies in DNA." J Biol Chem 266(23): 15160-9.

Doktycz, M. J., R. F. Goldstein, et al. (1992). "Studies of DNA dumbbells. I. Melting curves of 17 DNA dumbbells with different duplex stem sequences linked by T4 endloops: evaluation of the nearest-neighbor stacking interactions in DNA." Biopolymers 32(7): 849-64.

Gao, X., E. Gulari, et al. (2004). "In situ synthesis of oligonucleotide microarrays." Biopolymers 73(5): 579-96.

Gao, X., E. LeProust, et al. (2001). "A flexible light-directed DNA chip synthesis gated by deprotection using solution photogenerated acids." Nucleic Acids Res 29(22): 4744-50.

Gotoh, O. and Y. Tagashira (1981). "Stabilities of Nearest-Neighbor Doublets in Double-Helical DNA Determined by Fitting Calculated Melting Profiles to Observed Profiles." Biopolymers 20(5): 1033-1042.

LeProust, E., J. P. Pellois, et al. (2000). "Digital light-directed synthesis. A microarray platform that permits rapid reaction optimization on a combinatorial basis." J Comb Chem 2(4): 349-54.

Marmur, J. and P. Doty (1962). "Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature." J Mol Biol 5: 109-18.

Panjkovich, A. and F. Melo (2005). "Comparison of different melting temperature calculation methods for short DNA sequences." Bioinformatics 21(6): 711-22.

Rouzina, I. and V. A. Bloomfield (1999). "Heat capacity effects on the melting of DNA. 1. General aspects." Biophys J 77(6): 3242-51.

Rouzina, I. and V. A. Bloomfield (1999). "Heat capacity effects on the melting of DNA. 2. Analysis of nearest-neighbor base pair effects." Biophys J 77(6): 3252-5.

SantaLucia, J., Jr. (1998). "A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics." Proc Natl Acad Sci U S A 95(4): 1460-5.

SantaLucia, J., Jr., H. T. Allawi, et al. (1996). "Improved nearest-neighbor parameters for predicting DNA duplex stability." Biochemistry 35(11): 3555-62.

Sundaralingam, M. and P. K. Ponnuswamy (2004). "Stability of DNA duplexes with Watson-Crick base pairs: a predicted model." Biochemistry 43(51): 16467-76.

Vologodskii, A. V., B. R. Amirikyan, et al. (1984). "Allowance for heterogeneous stacking in the DNA helix-coil transition theory." J Biomol Struct Dyn 2(1): 131-48.

Wetmur, J. G. (1991). "DNA probes: applications of the principles of nucleic acid hybridization." Crit Rev Biochem Mol Biol 26(3-4): 227-59.

Wu, P., S. Nakano, et al. (2002). "Temperature dependence of thermodynamic properties for DNA/DNA and RNA/DNA duplex formation." Eur J Biochem 269(12): 2821-30.