Fluorescence quenching intercalation

In some cases it is possible to obtain a measure of the association constant for intercalation directly from fluorescence quenching data. This method is applicable when the dynamic quenching of the hydrocarbon fluorescence by DNA is small and when the intercalated hydrocarbon has a negligible fluorescence quantum yield compared to that of the free hydrocarbon. If these conditions are met, the association constant for intercalation, Kq, is equal to the Stern-Volmer quenching constant Kgy (76) and is given by Equation 1. 

DNA forms an intercalation product with the trypanoacids and ethidi-nium [178,179]. Olinsted and Kearns [180] studied the fluorescence quench-... 

There have been numerous investigations of the fluorescence quenching of intercalated ethidium (ET+ Table 5) by intercalated or groove bound quenchers [128-... 

An evanescent wave biosensor was devised for determination of analytes capable of intercalation in dsDNA in a FIA system. A polyethylene lensed optical fiber is coated with a thin polymeric layer containing dsDNA which is immobilized there. The fiber is placed in a FLA system immersed in a solution of ethidium bromide (144), which undergoes intercalation in the dsDNA. The fluorescence signal of 144 is thus enhanced about a 1000-fold relative to the evanescent wave fluorescence measurement without the coating and is dependent on the concentration in solution. If an analyte is present in the same solution, it competes with 144 for intercalation in the DNA and causes fluorescence quenching, which can be measured and correlated to the analyte concentration. This method was applied to determination of various analytes, including 4, 6-diamidino-2-phenylindole dihydrochloride (145)247. 

A series of DNA duplexes ranging from 10 to 14 base pairs in length were prepared with the intercalators attached to opposite 5 ends through short aliphatic linkers. Despite the long distances (17-35 A) that separated the intercalators, fluorescence quenching occurring on a subnanosecond timescale was detected [12]. 

Intercalation of ethidium bromide (EB ) into calf-thymus DNA (5 mM phosphate buffer, pH 7) occurs with a saturation of 0.4/base-pair and, at [EB ] = 67 nK and [DNA-bases] = 1.4 mM, more than 99% of EB will be intercalated. Assuming a nonrandom distribution, the average distance between chro-mophores is 10 base-pairs ( 34 A). Fluorescence from Intercalated EB decays with a lifetime (tq) of (21.1 0.1) ns, compared to (1.6 0.1) ns in water. Upon addition of N,N -dimethyl-2,7-diazapyrenium dichloride (DAP ), which intercalates with high affinity, fluorescence from EB was quenched and decay profiles became markedly non-exponential, even at very low concentrations of quencher. 

Electrochemical indication makes use either of redox labels, which are bound chemically to DNA strands, or of redox active intercalators . The latter are flat molecules able to intercalate the windings of DNA helix. It has been shown, that the well known, commercially available fluorescence quenching agent dabcyl (p-methyl red) is useful also for hybridisation detection with heated electrodes [51]. It was found that osmium tetroxide in different forms is a most powerful redox label for hybridisation detection, especially at heated electrodes [42, 52-55]. OSO4 has been used as bipyridyl complex and also botmd to a heated gold electrode surface via thiol bridges. 

The fluorescence of BaPT OS) and of other related compounds (40) seems to be nearly completely quenched upon physical intercalation into DNA. 

Since tRNA is more varied structurally than DNA, ethidium could reside in pockets as well as intercalate into double-strand regions. The fluorescence decay provides information about the type, or types, of binding sites occupied by ethidium. It is currently believed that the excited state of ethidium is quenched by proton transfer to the solvent0 86-1 and that its lifetime is reduced with increasing solvent exposure. If ethidium occupies two or more kinds of sites with different degrees of exposure to solvent, then its fluorescence decay is expected to be multiexponential. 

In contrast to the results obtained with EB, weak excitonic interactions in ACR intercalated at the BAZrP galleries result in broadened absorption spectrum only at high loadings (Fig. 23), suggesting that the intermolecular forces are relatively weak for this chromophore [55]. The fluorescence spectra of ACR intercalated into BAZrP show a large blue shift from 480 nm (free form) to 450 nm (bound form) (Fig. 24), and the fluorescence intensity also decreases monotonously with the concentration of ACR, ascribed to self-quenching. This blue shift of ACR fluorescence is also in contrast to the 20-nm red shift observed for EB bound to BAZrP. 

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