Ethidium bromide does not fluoresce when intercalated adjacent to 7-deazaguanine in duplex DNA.
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.
Latimer
L J
LJ
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Lee
J S
JS
D004247:DNA / Q000737:chemistry*; D004996:Ethidium / Q000737:chemistry*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D007364:Intercalating Agents; D009691:Nucleic Acid Denaturation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D007364:Intercalating Agents; D006147:Guanine; D004247:DNA; D004996:Ethidium; C066856:7-deazaguanine