Investigation of Förster resonance energy transfer (FRET) and competition of fluorescent dyes on DNA microparticles

Abstract

Fluorescent labeling is widely used to investigate the structural stability and changes to DNA nano- and microstructures. Despite this, the conventional method for observing DNA structures has several limitations in terms of cost-efficiency. This paper introduces a DNA spherical particle stained with DNA intercalating dyes (SYBR Green and SYTOX Orange) as tracers and reports the interaction between multiple dyes. The interference between the dyes was analyzed in terms of Förster resonance energy transfer (FRET) and competition. The changes in the fluorescence intensity by FRET were uniform, regardless of the sequence. The competition effect could occur when several dyes were added simultaneously. These properties are expected to help in the design of multicolor tracers in bioimaging and environmental applications.

Figure 1

Scanning electron microscopy (SEM) and fluorescent microscopy images of ssDNA balls (top panels) and dsDNA balls (bottom panels). (a) SEM was used to analyze the morphology of the DNA balls; (b) Fluorescent microscopy images of the DNA balls stained with SYBR Green and SYTOX Orange. Scale bars, 1 μm in (a) and 10 μm in (b).

Figure 2

Image cytometry acquisition plots of two-color analysis of the ssDNA balls stained with SYBR Green or SYTOX Orange. The scatter plots show the green fluorescence intensity versus the orange fluorescence intensity. SG = SYBR Green; SO = SYTOX Orange.

Figure 3

Image cytometry acquisition plots for two-color analysis of the dsDNA balls stained with SYBR Green or SYTOX Orange. The scatter plots show the green fluorescence intensity versus the orange fluorescence intensity. SG = SYBR Green; SO = SYTOX Orange.

Figure 4

Intensity change in the DNA balls. Each balls were stained with either SYBR Green or SYTOX Orange or both. The blue arrow indicates the decrease in green fluorescence intensity. (a) The ssDNA balls were stained with the fluorescent dyes; and (b) The dsDNA balls were stained with the fluorescent dyes.

Figure 5

Alteration of the FRET dependent on the staining methods. The dsDNA balls were stained with the green and orange dyes simultaneously or in series. In the serial staining, SYTOX Orange was added after SYBR green staining (a) SYBR Green I was used as the green fluorescent dye; (b) SYBR Green II was used as the green fluorescent dye; and (c) Decrease in the green fluorescence intensity depending on the staining methods.