Hybridization‐Sensitive On–Off DNA Probe: Application of the Exciton Coupling Effect to Effective Fluorescence Quenching

Abstract

The design of dyes that emit fluorescence only when they recognize the target molecule, that is, chemistry for the effective quenching of free dyes, must play a significant role in the development of the next generation of functional fluorescent dyes. On the basis of this concept, we designed a doubly fluorescence-labeled nucleoside. Two thiazole orange dyes were covalently linked to a single nucleotide in a DNA probe. An absorption band at approximately 480 nm appeared strongly when the probe was in a single-stranded state, whereas an absorption band at approximately 510 nm became predominant when the probe was hybridized with the complementary strand. The shift in the absorption bands shows the existence of an excitonic interaction caused by the formation of an H aggregate between dyes, and as a result, emission from the probe before hybridization was suppressed. Dissociation of aggregates by hybridization with the complementary strand resulted in the disruption of the excitonic interaction and strong emission from the hybrid. This clear change in fluorescence intensity that is dependent on hybridization is useful for visible gene analysis.

Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit

“…[30][31][32] To investigate the thermal stability of the DNA triplexes formed by the ECHO probes, thermal melting experiments of the DNA triplexes were performed and the absorbance at 260 nm of the DNA triplexes was monitored with increasing temperature. The melting curves of T 1 and T 2 in the presence of the D 1 /D 2 duplex at pH 6, 7 or 8 are shown in Figure 4A and B.…”

“…A similar phenomenon was observed for the fluorescent probes labeled with one or two molecules of TO. 32 Moreover, the labeling with the TO dimers at the two thymidine residues caused a problem in the determination of the dissociation temperature of T 5 . The melting curves derived from the absorption at 510 nm were not sigmoidal, which might be caused by the overlap of the subtly different two sigmoidal curves.…”

“…When the amount of the reported previously. 28,31,32 Purified DNA oligonucleotides labeled with TO were identified using MALDI-TOF mass spectrometry. "M" indicates 5-methylcytosine.…”

“…We have reported fluorescent oligonucleotide probes labeled with a TO dimer unit, which we call ECHO (exciton-controlled hybridization-sensitive fluorescent oligonucleotide) probes, for the detection of single-stranded DNA and RNA. [21][22][23][24][25][26][27][28][29][30][31][32] ECHO probes emitting low background fluorescence were expected to be also applicable to detect double-stranded DNA as TO molecules emit intense fluorescence in the presence of DNA triplexes. 15,17 However, DNA triplexes formed by ECHO probes have not been reported and the photophysical properties are unknown.…”

“…of a structural change in the TO groups from an H-dimer to monomeric units upon the hybridization with a DNA or RNA complementary strand, which was deduced from the red shift of the visible light absorption derived from the TO moiety. [30][31][32] Similarly, the wavelengths of the absorption maximum of the two TO molecules on the TFOs might be changed by the DNA triplex formation. To confirm this, the absorption spectra of T 2 , T 3 and T 4 in the absence and presence of a D 1 /D 2 duplex were measured in buffers at pH 6, 7 and 8.…”