Bulk and Single-Molecule Fluorescence Studies of the Saturation of the DNA Double Helix Using YOYO-3 Intercalator Dye

Abstract

We report a thorough photophysical characterization of the interactions between double-stranded DNA (dsDNA) and the trimethine cyanine homodimer dye YOYO-3. The fluorescence emission of this dye is enhanced by intercalation within the DNA double helix. We have explored the saturation of the dsDNA by bound YOYO-3 at the single-molecule level by studying the single-pair Förster resonance energy transfer (FRET) from an energy donor, Alexa Fluor 488, tagged at the 5' end of the double helix and the energy acceptor, YOYO-3, bound to the same DNA molecule. The spontaneous binding of YOYO-3 gives rise to an effective distribution of different FRET efficiencies and, therefore, donor-acceptor (D-A) distances. These distributions reveal the existence of multiple states of YOYO-3. Steady-state and time-resolved fluorescence and circular dichroism confirmed the presence of a DNA-bound aggregate of YOYO-3, conspicuous at high dye/base pair ratios. The spectral features of the aggregate suggest that it may have the structure of a parallel H-aggregate.

Optical determination of the electronic coupling and intercalation geometry of thiazole orange homodimer in DNA

“…S6, which is consistent with dynamic quenching observed in YOYO and cyanine dimers. 13,43 Together this suggests that aggregation of the TOTO occurs for high TOTO:DNA ratios, where several TOTO molecules may interact with the DNA, e.g., by groove binding or through multiple intercalated TOTO within the DNA base stack. Both cases could give rise to the observed evidence of strong coupling.…”

“…16,41 Significant non-intercalating populations of YOYO have also been observed at dye loading concentrations that are much higher than the 1:1 TOTO:dsDNA concentration used here. 42,43 A small but non-negligible fraction of nonintercalated TOTO may be responsible for the sub-population with low coupling strengths. To address this possibility, we consider a model that consists of two TOTO/dsDNA geometries: one that is fully bis-intercalated and exhibits moderate TO-TO coupling and a second component that has weaker TO-TO coupling, which might arise, e.g., from partially intercalated or groove bound TOTO.…”

“…Similar observations have been made for YOYO at high ratios of dye:bp. 42,43 For the lower TOTO:DNA ratios (i.e., 1:5, 1:2, 1:1), the slight increase in the vibronic peak intensity might be an indication of instances in which more than one TOTO binds to DNA. This is consistent with exciton-vibration coupling theory that predicts a redistribution of oscillator strength towards higher energy vibronic transitions as the electronic coupling increases.…”

“…Linear and circular dichroism studies suggest that the dominant geometry at these high dye:bp ratios is a groove bound dimer with nearly orthogonal dipoles. 42,43 By comparison, the anisotropy dynamics show only subtle changes with TOTO:dsDNA ratio, Fig. S8 of the supplementary material.…”

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

“…Most of the dyes also seem to interact with ssDNA and some, such as EtBr, have also been found to interact with RNA [91,92]. The combination of nucleic acid sensitive dyes and FRET has been used in a number of studies dealing with understanding how the dyes interact with DNA [92][93][94] and in the detection of single-nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) [95][96][97].…”