Pyrimidine bases, fluorescence

Daniels M, Hauswirth W (1971) Fluorescence of the purine and pyrimidine bases of the nucleic acids in neutral aqueous solution at 300 K. Science 171 675... 

In general, reduction potentials of nucleobases have been studied much less than their oxidation potentials, and in particular water-based data are rather lacking [2, 35]. We therefore listed the available polarographic potentials measured in dimethylformamide and data obtained from pulse radiolysis studies or fluorescence quenching measurements. From the data in Table 1, it is evident that the pyrimidine bases are most easily reduced. The reduction potential of the T=T CPD lesion is close to the estimated value of the undamaged thymine base [34, 36]. 

The close correspondence of the DNA absorption spectrum with that of a mixture of mononucleotides of the same composition illustrates the weak nature of the interactions between neighboring purine and pyrimidine bases guanine (G), cytosine (C), adenine (A), and thymine (T) at an interplanar separation of 3.36 A in the unexcited double-helical configuration. On the other hand the structureless fluorescence band of (calf-thymus) DNA is red-shifted by 3500 cm-1 from the fluorescence spectral origin of the mononucleotides it closely resembles the fluorescence spectrum of the dinocleotide ApT (and of poly dAT) and is accordingly identified131 with the fluorescence... 

Fluorescence assays are considered among the most convenient, sensitive, and versatile of all laboratory techniques. However, the purine and pyrimidine bases yield only weak fluorescence spectra. Le Pecq and Paoletti (1967) showed that the fluorescence of a dye, ethidium bromide, is enhanced about 25-fold when it interacts with DNA. Ethidium bromide, which is a relatively small planar molecule (Figure El3.4), binds to DNA by insertion between stacked base pairs (intercalation). The process of intercalation is especially significant for aromatic dyes, antibiotics, and other drugs. Some dyes, when intercalated into DNA, show an enhanced fluorescence that can be used to detect DNA molecules after gel electrophoresis measurements (see Chapter 4 and Experiments 14 and 15) and to characterize the physical structure of DNA. Two analyses of DNA will be completed in this experiment ... 

Experimentally, the direction of a transition moment in a molecule can he evaluated by four methods (i) polarized spectra of single crystals, (ii) fluorescence or phosphorescence polarization, (iii) spectra of molecules embedded in stretched Aims, and (iv) spectra of molecules oriented by external fields. Only relative directions of the transition moment can be determined by means of the last three methods, whereas the polarized spectra of single crystals give the absolute direction of the moments if the crystal structure is known. The first method has been applied to the study of the electronic structure and spectra of several pyrimidine bases of nucleic... 

Fluorescence polarization data for the binding of RNase A to Fl-d(UAA), Fl-d(AAA) and Fl-d(0AA) are shown in Figure 3. RNase A binds Fl-d(UAA) approximately 20-fold more tightly than n-d(AAA) or Fl-d(0AA), demonstrating that the B1 subsite has affinity for a pyrimidine base. The similarity in binding affinity for Fl-d(AAA) and Fl-d(0AA) indicates that the B1 subsite of RNase A does not bind adenine significantly, but does not discriminate against it. 

It is generally believed that among the pyrimidine bases, uracil and thymine mainly exist in the keto form. However, the existence of a small amount of the enol form of these compounds has also been indicated in some experiments. Hauswirth and Daniels have explained the observed deviation in the excitation spectrum of thymine firom the corresponding absorption spectrum in terms of the possibility of emission firom the enol tautomer. On the other hand, Vigny and Duquesne observed that both absorption and fluorescence excitation spectra of thymine are in resemblance with each other. Suwaiyan et al. have suggested the existence of a small amount of the enol tautomer in aqueous solutions of 5-chlorouracil at room temperature. The existence of keto-enol tautomerism in uracil, thymine and their derivatives has also been suggested in supersonic jet-cooled spectroscopic studies. ... 

A modification of Sanger s method has resulted in the commercial availability of automated DNA sequenators based on Sanger s use of dideoxy analogs of nucleotides. Instead, however, of tagging a primer with P, the purine and pyrimidine base portions of the dideoxynucleotides are each modified to contain a side chain that bears a different fluorescent dye, and all the dideoxy analogs are present in the same reaction. After electrophoretic separation of the products in a single lane, the gel is... 

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