Unstacked bases

Stacked bases in nucleic acids absorb less ultraviolet light than do unstacked bases, an effect called hypochromism. Thus, the melting of nucleic acids is easily followed by monitoring their absorption of light, which peaks at a wavelength of 260 nm (Figure 517). 

Hehcal segments are interspersed with bulge, internal and hairpin loops that contain noncanonical base pairs and unstacked bases. 

Fig. 3. The X-ray crystal structure of Sac7d-d (GTAATTAC)2 complex determined to 1.9 A resolution. The W23 side chain is shown at the protein-DNA interface. The intercalating valine and methionine are not shown for clarity, but lie in the pocket above W23 between unstacked base pairs 3 and 4 (PDB accession codes lazq and lazp). Prepared with MIDAS [T. Feirin, C. Huang, L. Jarvis, and R. Langridge, Mol. Graphics 6,13 (1988)].

For example, the temperature-induced transition between native and random coil states of a nucleic acid can be conveniently monitored by UV spectrophotometry. The reason for this is that stacked bases have a lower absorbance per base than unstacked bases this is called hypochromicity, which is defined as ... 

The central 10 base pairs of the palindromic DNA molecule have a regular B-DNA structure. Between base pairs 5 and 6 in each half of the fragment (base pairs are counted from the center) there is a 40° kink which causes these base pairs to be unstacked (Figure 8.24a). After this localized kink the two end regions have an essentially B-DNA structure. The kink occurs at a TG step in the sequence GTG. These TG steps at positions 5 and 6 are highly conserved in both halves of different CAP-binding sites, presumably in part because they facilitate kinking. 

The double-stranded structure of DNA can be separated into two component strands (melted) in solution by increasing the temperature or decreasing the salt concentration. Not only do the two stacks of bases puU apart but the bases themselves unstack while still connected in the polymer by the phosphodiester backbone. Concomitant with this denaturation of the DNA molecule is an increase in the optical absorbance of the purine and pyrimidine bases—a phenomenon referred to as hyperchromicity of denaturation. Because of the... 

Wu P, Norland TM, Gildea B, McLaughlin LW (1990) Base stacking and unstacking as determined from a DNA decamer containing a fluorescent base. Biochem 29 6508-6514... 

Fig. 3 Thermal denaturation transition of a DNA helix, (a) UV absorbance increases with temperature, following the unstacking of bases, following a sigmoidal shape. AD and Au are lower and upper baselines, also slightly dependent on temperature, (b) Fraction of single strands 6 extracted from data in (a), which defines the melting temperature corresponding to 9 = 0.5. Adapted with permission from [7]...

Mandal, S. S., Fidalgo da Silva, E., and Reha-Krantz, L. J. (2002). Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase. Biochemistry 41, 4399—4406. 

While denaturation can be detected readily through changes in viscosity, a much more convenient way to detect it is by ultraviolet (uv) absorption measurement. The difference in the uv absorption spectra of the native (double-helical) and denatured (single-stranded) forms of DNA is shown in Fig. 7-7. At the wavelength of maximum absorption (260 nm), absorption by single-stranded DNA is approximately 40 percent higher than by double-stranded DNA. This is referred to as the hyperchromic effect and results from the unstacking of the base pairs in the helix. 

Continuous buffer systems have been almost universally superseded by disctoninuous buffers for SDS-PAGE. The most popular system (31) is based on the Ornstein-Davis system (2,3) with the addition of 5PS. It should be noted that the nature of sTa ing is modified in the presence of SDS and it is not necessary to have a discontinuity in pH as unstacking will occur by the change in gel concentration alone ( ). 

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