WEB-THERMODYN is a web-based program that computes the thermodynamic profile of duplex DNA structures. It requires the input values of temperature (°C), salt concentration or monovalent cation concentration (mM), name of the DNA molecule, the base sequence of the DNA of interest, the shape of the DNA (linear or circular), the analysis window size, the step size of "sliding" and the number of marked windows of minimal free energy values.

The program employs a sliding-window-analysis approach to dissect the thermodynamic profile of a DNA sequence. See the following illustration:

The free energy values(G) of these "sliding windows" are computed using the Nearest-Neighbor-Frequency Algorithm described below. The start positions of these windows and the corresponding free energy values are listed in a table. In the right columns of the table symbolic illustrations of the relative helical stability of the window according to the G values are shown. The same data is also output to a ASCII txt file for the convenience of further data analysis using other software such as SLIDEWRITEPLUS and MICROSOFT EXCEL. Other output of the program includes the name of the DNA molecule and input paramenters by the user such as sequence, shape, window size, step size, no. of marks, temperature, salt concentration, etc. Also output are the length of the molecule, number of G or C bases, GC composition (GC%). Similar PC-based software, THERMODYN, was developed and described by Natale and Kowalski. (Natale et al, 1992; Natale et al, 1993)

The principles upon which the Nearest-Neighbor-Frequency algorithm is based are:

Under a given set of solution conditions the relative stability of a DNA duplex structure depends on the primary sequence. More specifically, the stability of a DNA duplex depends primarily on the identity of the nearest-neighbor (NN) bases.
 

Ten different nearest-neighbor (NN) interactions are possible in a Watson-Crick DNA duplex structure: AA/TT; AT/TA; TA/AT; CA/GT; GT/CA; CT/GA; GA/CT; CG/GC; GC/CG; GG/CC. The standard (under conditions of 25°C and 1M monovalent cation) entropy (S°) and standard enthalpy (H°) values of all of these 10 interactions are available from two different DATA SETS. The CURRENT data set is the unified data set from SantaLucia, J., Jr. (1998). The PREVIOUS data set from Breslauer et al. (1986) is provided for back-compatibility with THERMODYN and an earlier version of WEB-THERMODYN.
 

The standard entropy value (S°) of a duplex DNA structure is determined by the sum of the standard entropy values of all of its nearest neighbors. Similary, the standard enthalpy (H°) of a duplex DNA structure is determined by the sum of the standard enthalpy values of all of its nearest neighbors.

H° = SUM {(frequency of NN)*(H° of NN)}

S° = SUM {(frequency of NN)*(S° of NN)}

The melting temperature of the duplex can then be calculated by the following formula once we have determined the S° and H°.

Tm = (H°/S°) + 18log[monovalent cation]

Note that the Tm of interest to us is for local strand separation within a larger duplex DNA and is therefore independent of DNA concentration.

Subsequently, the free energy value (G) of the duplex can be calculated by the following formula:

G =H°[1-(T/Tm)]