Abstract
Several synthetic DNAs were prepared containing the unusual bases 7-deazaadenine (c7A) and 7-deazaguanine (c7G). As judged from changes in melting temperatures these modified DNAs bound ethidium to a similar extent as the parent polymers. However, duplexes such as poly [d(Tc7G)].poly[d(CA)] and poly[d-(TC)].poly[d(c7GA]) gave no enhancement of ethidium fluorescence in a standard ethidium fluorescence assay. Fluorescence spectra in the range 400-650 nm showed that ethidium bound to poly[d(TC)].poly[d(Gc7A)] gave 70% of the fluorescence of the parent polymer poly[d(TC)].poly[d(GA)], whereas the fluorescence of poly[d(TC)].poly[d(c7GA)] was essentially 0%. Even the intrinsic fluorescence of ethidium in solution was quenched in the presence of poly[d(TC)].poly[d(c7GA)]. Binding constants were estimated from Scatchard analysis and were 4.8, 3.4, and 2.0 x 10(6) M-1 for poly[d(TC)].poly[d(GA)], poly[d(TC)].poly[d(Gc7A)], and poly[d(TC)].poly[d(c7GA)], respectively. This reduction in binding constant cannot account for the loss of fluorescence. The UV spectrum of ethidium was measured in the presence of these DNAs, and some significant differences were noted. Presumably the presence of 7-deazaguanine alters the electronic structure of bound ethidium so that it can no longer fluoresce.
Highlights
Several synthetic DNAs were prepared containing The molecular basis for the fluorescence enhancement has the unusual bases 7-deazaadenine (c’A) and 7-deaza- been postulated to be due to anincrease in the energy sepaguanine (c’G)
Fluorescence in a standard ethidium fluorescence as- This model is attractive since proton acceptors quench the say
The ethidiumis less accessible to solvent in the intercalated state; similar reasonimngay explainwhy the triplex poly(dT) -poly(dA) -poly(gdiTve)s greater fluorescence enhancement than theduplex poly(&) .poly(dT) [11]
Summary
Ture ofbound ethidium so that it can no longer fluoresce. The bindingof ethidium to DNA has been studied foovrer 25 years [1, 2].Details of the interaction arewell understood, sincethestructure of ethidium-DNA complexes hasbeen reported at high resolution by x-ray crystallography [3, 4]. For the poly[d(TC)] .poly[d(GA)] family, accurate binding parameters were measured from Scatchard plots asshown in Fig. 3.The binding constant, K, is significantly lower for both the duplexes containing deazapurines, but simple calculations show that thelevel of ethidium binding under the conditions of the fluorescence assay is scarcely altered. The possibility that c7G somehow accelerates proton transfer from excited state ethidium to solvent appears unlikely, since the pK, for c7Gis higher than for guanine itself (10.3 compared with pK, values of 4.5 and 9.3 for guanine) [21] In this regard the significant fluorescencegivenby poly[d(TTC)].poly[d(c7GAA)]is important. The quenching of fluorescence by c7Gwas most unexpected but serves as areminder that optical properties are exquisitely sensitive to theenvironment of the chromophore
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