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Chemistry and Biology of Non-canonical Nucleic Acids. Naoki Sugimoto
Читать онлайн.Название Chemistry and Biology of Non-canonical Nucleic Acids
Год выпуска 0
isbn 9783527817863
Автор произведения Naoki Sugimoto
Жанр Химия
Издательство John Wiley & Sons Limited
(3.5)
(3.6)
For a unimolecular transition,
where
Figure 3.4 shows a UV melting curve for the self-complementary duplex (5′-ATGCGCAT-3′). At low temperatures, the strands are in duplex form and the absorbance is low. As the temperature is increased, the duplex dissociates into single strands. The UV absorbance of the duplex is increased by dissociation of the duplex, and the increment of absorbance is referred to as hyperchromicity. (The opposite, a decrement of absorbance, is called hypochromicity.) For self-complementary or non-self-complementary duplexes with equal concentrations of each strand, the melting temperature, Tm (in degrees Kelvin), is the point at which the concentrations of strands in duplex and in single strands are equal (Figure 3.4a). The steepness of the transition indicates the cooperativity of the transition. The width and maximum of the first derivative of the melting curve can also indicate the cooperativity and melting temperature, although the peak of the derivative curve only occurs at the Tm only when the transition is unimolecular [5]. Tm is most accurately measured by fitting the lower and upper baselines. The melting temperature is measured at several concentrations over a 100-fold range and then plotted versus the concentration in a van't Hoff plot. The van't Hoff equation relates the Tm (in degrees Kelvin), Ct, ΔH°, and ΔS°:
(3.9)
where R is the ideal gas constant, 1.987 cal K−1 mol−1 or 8.314 J K−1 mol−1. The slope of the van't Hoff plot gives the ΔH°, and the y-intercept gives the ratio of ΔH° to ΔS°. The free energy and equilibrium constant at any temperature can then be calculated using Gibb's relation:
(3.10)
To increase the accuracy of these parameters, data analysis can be performed by curve fitting as shown below. When the ratio of the double-stranded DNA is represented by α, absorbance (A) at a temperature (T) is calculated as
where εds and εss indicate the absorbance for the single-stranded and double-stranded DNA, respectively, and l and Ct represent the length of the light pass (or the path length of the cuvette used) and the total concentration of DNA strands, respectively. The εds, εss, and observed equilibrium constant (Kobs) for the duplex formation can be represented as follows with the assumption that absorbance is directly proportional to temperature:
where R is the gas constant and mds and bds or mss and bss represent the slope and intercept of the upper baseline or lower baseline for the melting curve of a duplex dissociation (Figure 3.4b), respectively. The six variables (εds, εss, bds, bss, ΔH°, and ΔS°) can be calculated by the curve fitting using Eqs. (3.11, 3.12, 3.13, 3.14). These calculations, which are performed using a PC equipped with the curve fitting software such as IGOR Pro, KaleidaGraph, or ORIGIN, derive the thermodynamic parameters from the shape of the melting curve.
Estimated errors in the thermodynamic values (σΔH°, σΔS°, and