We have developed a new quencher-free stemless linear probe involving multiple perylenes incorporated through D-threoninol; each perylene is separated by intervening natural nucleotides. Without a substrate, the flexible linear probe does not emit fluorescence due to the self-quenching of the weakly interacting fluorophores. Upon hybridization with the target, intercalation of each dye between the base pairs results in emission of strong fluorescence. The maximum signal-background ratio attained was 180, and the response rate was significantly faster than that of a classic hairpin-forming molecular beacon.

In the presence of poly(L-lysine)-graft-dextran, an in-stem molecular beacon involving three perylene-anthraquinone pairs in the stem region had a signal/background ratio of as high as 570. Response speed was also remarkable; equilibrium was attained within 5 minutes after addition of substrate DNA at 20 °C.

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Asymmetric dye clusters with a single fluorophore (Cy3) and multiple quenchers (4'-methylthioazobenzene-4-carboxylate, methyl red, and 4'-dimethylamino-2-nitroazobenzene-4-carboxylate) were prepared. The dye and one-to-five quenchers were tethered through D-threoninol to opposite strands of a DNA duplex. NMR analysis revealed that the clusters with a single fluorophore and two quenchers formed a sandwich-like structure (antiparallel H-aggregates). The melting temperatures of all the heteroclusters were almost the same, although structural distortion should become larger, as the number of quenchers increased. An asymmetric heterocluster of a single fluorophore and two quenchers showed larger excitonic interaction (i.e., hypochromicity of Cy3), than did a single Cy3 and a single quencher. Due to the larger exciton coupling between the dyes, the 1:2 heterocluster suppressed the background emission more efficiently than the 1:1 cluster. However, more quenchers did not enhance quenching efficiency due to the saturation of exciton coupling with two quenchers. Finally, this asymmetric 1:2 heterocluster was introduced into the stem region of a molecular beacon (MB; also known as an in-stem MB) targeting the fusion site in the L6 BCR-ABL fusion gene. With this MB design, the signal/background ratio was as high as 68 due to efficient suppression of background emission resulting from the maximized excitonic interaction.