Probing the Interaction Between Fluorophores and DNA Nucleotides by Fluorescence Correlation Spectroscopy and Fluorescence Quenching^†

Abstract

We have investigated the association interactions between the fluorescent dyes TAMRA, Cy3B and Alexa-546 and the DNA deoxynucleoside monophosphates by means of fluorescence quenching and fluorescence correlation spectroscopy (FCS). The interactions of Cy3B and TAMRA with the nucleotides produce a decrease in the apparent diffusion coefficient of the dyes, which result in a shift toward longer times in the FCS autocorrelation decays. Our results with Cy3B demonstrate the existence of Cy3B-nucleotide interactions that do not affect the fluorescence intensity or lifetime of the dye significantly. The same is true for TAMRA in the presence of dAMP, dCMP and dTMP. In contrast, the diffusion coefficient of Alexa 546 remains practically unchanged even at high concentrations of nucleotide. These results demonstrate that interactions between this dye and the four dNMPs are not significant. The presence of the negatively charged sulfonates and the bulky chlorine atoms in the phenyl group of Alexa 546 possibly prevent strong interactions that are otherwise possible for TAMRA. The characterization of dye-DNA interactions is important in biophysical research because they play an important role in the interpretation of energy transfer experiments, and because they can potentially affect the structure and dynamics of the DNA.

CTAB enhancement of FRET in DNA structures

“…The emission intensity of TAMRA on the dsDNA (strand Ia + II) was much higher compared to TAMRA on ssDNA in absence of CTAB (Figure a). Similar to previous studies, the emission intensity of TAMRA conjugated on the ssDNA was quenched by free guanine bases in DNA . Therefore, hybridization reduces the quenching by the free bases and recovers the fluorescent emission from the TAMRA.…”

“…It is hypothesized that the difference might be attributed to the different sequences of the conjugated DNA and the material properties of dye such as hydrophobicity and quenching sensitivity by DNA bases. For example, since it is known that the fluorescence emission of TAMRA is more susceptible to be quenched by a guanine base , the emission intensity of TAMRA conjugated on strand I, which is already quenched due to DNA bases, could recover the original emission by hybridization and CTAB aggregation.…”

“…Additionally, it has been reported in other studies, that electron‐transfer from a DNA base to the excited singlet state of a dye can also quench the fluorescence emission . This quenching is most efficient for guanine, which has the lowest oxidation potential among the four DNA bases . Other common contact quenching, also referred to as static quenching, can occur when dyes are aggregated due to hydrophobic effects in which the dye molecules stack together to minimize contact with water .…”

Manganese‐Induced Triplet Blinking and Photobleaching of Single Molecule Cyanine Dyes

“…in which 〈 N 〉 represents the mean number of dye molecules within the observation volume, D is the diffusion coefficient of the dye, r 0 and z 0 are the radial and axial semi‐axis of the observation volume, respectively, T is the steady‐state fraction of molecules in the triplet state, and τ T is the triplet relaxation time 15c. To fit the experimental data, the diffusion coefficient of Cy3B was taken as 440 μm 2 s −1 ,16 and the value of z 0 was taken as 10 r 0 . Note that the precise knowledge of the value of z 0 is not critical because z 0 ≫ r 0 , which minimizes the contribution of the term containing the parameter z 0 .…”

Study on the Interaction between Quinine Sulfate and DNA by Multiple Spectral Methods and Their Analytical Applications

“…[4][5][6] Fluorescence spectrometry is an extremely easy and useful tool for studying the interactions between small molecules and DNA. [7][8][9][10] The changes of the macromolecules in the physical and chemical environment as well as the dynamic properties after the interaction with small molecules can be detected by fluorescence spectrometry.…”