Nucleic acid melting temperature

A plot of the optical absorbance at 260 nm (the wavelength of maximum light absorption by nucleic acids) versus temperature is known as a melting curve (Fig. 5-45). The absorbance is lower, by up to 40%, for native than for denatured nucleic acids. This hypochromic effect (Chapter 23) is a result of the interaction between the closely stacked bases in the helices of the native molecules. The melting temperature Tm is taken as the midpoint of the increase in absorbance (Fig. 5-45). As the percentage of G + C increases, the nucleic acid becomes more stable toward denaturation because of the three hydrogen bonds in each GC pair. Tm increases almost linearly with increases in the G + C content. In the "standard" citrate buffer (0.15 M NaCl + 0.015 M sodium citrate, pH 7.0) Eq. 5-22 holds. The exact numerical relationship depends strongly upon the ionic composition and pH of the medium.37 72 552 553... 

Applications to fluorescent or fluorescently labeled proteins and nucleic acids, and to fluorescent lipid probes in phospholipid bilayers, have been reported. In the latter case, the diffusion coefficients measured above the chain melting temperature were found to be 10 7 cm2 s 1, which is in agreement with values obtained by other techniques. 

In 2004, Rayner and coworkers reported a dynamic system for stabilizing nucleic acid duplexes by covalently appending small molecules [34]. These experiments started with a system in which 2-amino-2 -deoxyuridine (U-NH ) was site-specifically incorporated into nucleic acid strands via chemical synthesis. In the first example, U-NH was incorporated at the 3 end of the self-complementary U(-NH2)GCGCA DNA. This reactive amine-functionalized uridine was then allowed to undergo imine formation with a series of aldehydes (Ra-Rc), and aldehyde appendages that stabilize the DNA preferentially formed in the dynamic system. Upon equilibration and analysis, it was found that the double-stranded DNA modified with nalidixic aldehyde Rc at both U-NH positions was amplified 34% at the expense of Ra and Rb (Fig. 3.16). The Rc-appended DNA stabilizing modification corresponded to a 33% increase in (melting temperature). Furthermore, imine reduction of the stabilized DNA complex with NaCNBH, resulted in a 57% increase in T. ... 

Melting Temperature. The double helix of polynucleotides described above becomes thermodynamically unstable at particular temperatures (with specified conditions of solute concentration, pH, etc.) and is transformed into the open random-coil arrangement. This transformation is rather sharp, and can be measured by the concurrent changes in a number of physical properties of the nucleic acid, such as the optical absorption coefficient. The midpoint of the transition region is called the melting point. 

Like proteins, nucleic acids can undergo denatur-ation. The strands of the double helix of DNA are separated and the double-stranded regions of RNA molecules "melt." Denaturation can be accomplished by addition of acids, bases, and alcohols or by removal of stabilizing counter ions such as Mg2+. The product is a random coil and denaturation can be described as a helix —> coil transition. Denaturation of nucleic acids by heat, like that of proteins, is cooperative (Chapter 7, Section A,3) and can be described by a characteristic melting temperature. 

Both the denaturation process in proteins and the melting transition (also referred to as the helix-to-coil transition) in nucleic acids have been modeled as a two-state transition, often referred to as the all-or-none or cooperative model. That is, the protein exists either in a completely folded or completely unfolded state, and the nucleic acid exists either as a fully ordered duplex or a fully dissociated monoplex. In both systems, the conformational flexibility, particularly in the high-temperature form, is great, so that numerous microstates associated with different conformers of the biopolymer are expected. However, the distinctions between the microstates are ignored and only the macrostates described earlier are considered. For small globular proteins and for some nucleic acid dissociation processes,11 the equilibrium between the two states can be represented as... 

Melting Transition Typical 360 MHz proton NMR spectra of proflavine poly(dA-dT) complexes, Nuc/D = 24 and Nuc/D = 8, in 1 M NaCl solution at temperatures below the midpoint for the dissociation of the complex are presented in Figures 17A and B respectively. The stronger base and sugar resonances can be readily resolved from the weaker proflavine resonances (designated by asterisks) in the presence of excess nucleic acid (Figure 17) so that the resonances of the synthetic DNA and the mutagen can be monitored independently of each other. 

The melting transition of the daunomycin poly(dA-dT) complex can also be monitored at the nucleic acid resonance line widths and the data for the adenosine H-8 resonance are plotted in Figure 28. The resonance is very broad at temperatures below the melting transition of the complexes (dashed curves in Figure 28) indicative of stiffening of the synthetic DNA by the bound anthracycline ring. 

At room temperature, these molecules occupy well-defined locations in their respective crystal lattices. However, they tumble freely and isotropically (equally in all directions) in place at their lattice positions. As a result, their solid phase NMR spectra show features highly reminiscent of liquids. We will see an illustration of this point shortly. Other molecules may reorient anisotropically (as in solid benzene). Polymer segmental motions in the melt may cause rapid reorientation about the chain axis but only relatively slow reorientation of the chain axes themselves. Large molecular aggregates in solution (such as surfactant micelles or protein complexes or nucleic acids) may appear to have solidlike spectra if their tumbling rates are sufficiently slow. There are numerous other instances in which our macroscopic motions of solid and liquid may be at odds with the molecular dynamics. Nuclear magnetic resonance is one of the foremost ways of investigating these situations. 

Metal ion-binding modes, that compete with H-bonds for the donor and acceptor atoms of nucleic acids, normally destabilize the base pairs or prevent their formation, and thus promote melting (dissociation) of DNA at elevated temperatures. However, some of these metal ions, for example, Cu +, Mn + and AP+, can also promote renaturation of the dissociated DNA strands upon cooling presumably via cross-linking between complementary polynucleotide strands. ... 

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