Self-quenching reporter molecules

Fig. 2 Design and reaction of self-quenching reporter molecules (SQRM). (a) To exploit the traditional stem-loop structure of the SQRMs, a computer algorithm searches an entire sequence of mRNA for complementary sequences of a desired length (X, stems) that are separated by a proscribed distance (Y, loop), (b) The complementary sequences are synthesized as SQRM possessing 5 -fluorescein and 3 DABCYL groups. In the absence of target, quenching of fluorescence occurs. Once hybridization of the loop sequence to a complementary target takes place, the moieties are separated and fluorescence can be detected [78]. Image reprinted by the permission from Oxford University Press...

Pattanayak V, Gifford LK, Lu P, Gewirtz AM (2008) Observed versus predicted structure of fluorescent self-quenching reporter molecules (SQRM) caveats with respect to the use of "stem-loop" oligonucleotides as probes for mRNA folding. RNA 14 657-665... 

In Figure 5, the normalized emission spectra of the two solid hybrid materials, GFP/SBA-15 and GFP/Aerosil , are reported. The shape of the emission profile for GFP/SBA-15 follows closely that of the GFP in buffer solution, whereas the photoemission intensity of GFP/Aerosil is one order of magnitude lower and slightly different in its tale shape (spectra at the actual intensities not reported). This reduction in intensity could be explained by a multilayer arrangement of the protein molecules on the amorphous nanoparticles, which would explain both the difference in emission spectra ("self-quenching effect") and the difference in adsorption amount shown above. 

It is very important to distinguish between self-quenching of the triplet state by a second triplet molecule, i.e., the process discussed in the previous section which is diffusion controlled, and self-quenching by ground-state molecules which is often absent or very small.70 However, this is not always the case, e.g., for chlorophyll a in benzene a ground-state quenching constant of 5 X 107 1.moles-1 sec.-1 has been reported.49... 

MV + results in poor quenching by the latter compared with the quenching efficiency in homogeneous solutions. Co-intercalation of photoinactive Zn(bpy)3 + results in decreased self-quenching due to the random distribution of the luminescent molecules and the inactive molecules [153. Also, whereas neutral propylviologen sulfonate (PVS) was found to be an effective quencher, MV + was not [153]. Studies with the aim of understanding racemic interactions between the optically active isomers of Ru(bpy)3-+ have also been reported [156-158]. It may be noted that only 50 % intercalation of Ru(bpy)3 + occurs when the reactant contains only one isomer, whereas 100 % intercalation proceeds for racemic mixtures. Intercalation with a sequential arrangement of A- and A-isomers has been proposed for this observation [161, 162]. 

In an article which is critical of many generally accepted molecular fluorescence parameters of aromatic molecules (and by inference the parameters for other systems), Birks emphasizes the precautions necessary to eliminate errors due to self-absorption secondary fluorescence and/or self-quenching.1 The points are made that reliable data for rf and Of are available for only a few compounds, e.g. diphenylanthracene (DPA), perylene, quinine bisulphate, and acridone, and that these provide suitable standards. The value of Of (DPA) is now set at 0.83. The importance of solvent effects on Of and t( of DPA is stressed in a publication which reports Tf for DPA in cyclohexane and benzene.2 The value of 6.95 0.04 ns for benzene solution is in good agreement with the earlier work of Birks and Dyson3 and Ware and Baldwin 4 7 (7.35 0.05 ns). The value obtained for cyclohexane solution, 7.58 0.04 ns, although in poor agreement with earlier results, is probably the most acceptable. The absolute fluorescence quantum yield of quinine bisulphate has also been redetermined (Of = 0.56).8... 

Several papers reported on the spatial determination of photoactive molecule in organized assemblies (76-79), and Pallavicini and coworkers reviewed on the use of luminescence as probe in self-assembly of multicomponent fluorescent sensors (80). Also, luminescence quenching studies on [Rufbpyls] in sodiiun dodecyl sulfate (SDS) micelles and hemimicelles by using a variety of quenchers were reported by Tiuro and coworkers (81) and then reviewed by De Schryver and coworkers (82). 

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