Bulge‐like Asymmetric Heterodye Clustering in DNA Duplex Results in Efficient Quenching of Background Emission Based on the Maximized Excitonic Interaction

Abstract

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.

Design of molecular beacons: 3′ couple quenchers improve fluorogenic properties of a probe in real-time PCR assay

“…Efficient quenching of the Cy3 dye with a couple of azobenzenebased dyes (methyl red, 4 0 -methylthioazobenzene-4-carboxylate or 4 0 -dimethylamino-2-nitroazobenzene-4-carboxylate) in the MB stem should also be noted. 13 Copper catalyzed azide-alkyne cycloaddition reaction (CuAAC, Huisgen-Meldal-Sharpless reaction, 'click chemistry') was reported 14 in 2002 and shortly thereaer, was applied for the attachment of a uorescent dye to an oligonucleotide. 15 The technique became a powerful tool for nucleic acid modication.…”

De NovoDesign of Functional Oligonucleotides with Acyclic Scaffolds

“…In this structure, monomer emission is quenched, and exciplex emission with a longer wavelength appears. In order to minimize background emission in the absence of the target, the ISMB ( FL‐MB ) was designed with doubled quenchers opposite the fluorophore …”