Fluorescent dyes are widely used for staining and visualization of DNA in optical microscopy based methods. Even though for some dyes the mechanism of binding is known, how this binding affects DNA remains poorly understood. Here we present a novel experimental study of the influence of staining dyes on DNA properties. We use atomic force microscopy which allows quantification and measurement of structural properties of stained DNA with nanometer resolution. We studied the influence of dyes on the persistence length, the total contour length, and the morphology of individual DNA molecules. We tested three widely used dyes known to differently bind DNA molecules, namely PicoGreen, Dapi, and DRAQ5. Based on our measurements, when imaged at typical concentrations (manufacturer suggested concentrations used for cell imaging), PicoGreen dye showed little effect, Dapi dye decreased the DNA persistence length, and DRAQ5 decreased the persistence length and elongated the DNA. When used at high concentrations, all of the dyes induced drastic changes in the DNA morphology. Our study clearly shows that DNA-binding dyes, irrespective of their DNA binding mechanisms, strongly influence the physical properties of DNA. These changes are strongly dose and dye type dependent and therefore should be taken into consideration when conducting experiments with DNA. ■ INTRODUCTIONFluorescence microscopy is a widely used technique in cell biology, biochemistry, and medicine among other fields of scientific research. The need to label and visualize DNA and chromatin is gaining importance in different fields of biological research, with experiments ranging from measurements on isolated DNA 1−6,11−13 to experiments in fixed cells and live cells. [7][8][9][10]14 There is a large variety of DNA labeling dyes: some binding to the minor/major grooves of double-stranded DNA, others intercalating into DNA, and some doing both. 1,8−20 The influence of DNA labeling dyes on the DNA structure and its properties is still a topic of debate, since no clear data exist on how strongly they influence physical properties. 11−19 Nevertheless, several works revealed that parameters such as the contour length and the persistence length of bare DNA can be significantly changed by fluorescent dyes upon binding. 11−20On the example of YOYO-1 dyes, Gunther et al. showed that the intercalation of the dye in double-stranded DNA modified its mechanical and structural properties. 11 Different studies showed that upon binding of YOYO dye the contour length of the DNA increased roughly linearly, reaching 30−40% elongation compared to bare DNA. In experiments performed with optical tweezers Sischka et al. compared different DNA binding agents with different binding modes (such as ethidium bromide, YO-1, distamycin-A, and YOYO), revealing changes in the mechanical response of DNA pulling as the function of the corresponding dye binding mechanisms. 16 Wojcik et al. showed that DRAQ5 dye detached H1 and H2B histones in a concentration dependent manner in live cells. 14 Hig...

Structural differentiation of bacterial chromatin depends on cooperative binding of abundant nucleoid-associated proteins at numerous genomic DNA sites and stabilization of distinct long-range nucleoprotein structures. Histone-like nucleoid-structuring protein (H-NS) is an abundant DNA-bridging, nucleoid-associated protein that binds to an AT-rich conserved DNA sequence motif and regulates both the shape and the genetic expression of the bacterial chromosome. Although there is ample evidence that the mode of H-NS binding depends on environmental conditions, the role of the spatial organization of H-NS-binding sequences in the assembly of long-range nucleoprotein structures remains unknown. In this study, by using high-resolution atomic force microscopy combined with biochemical assays, we explored the formation of H-NS nucleoprotein complexes on circular DNA molecules having different arrangements of identical sequences containing high-affinity H-NS-binding sites. We provide the first experimental evidence that variable sequence arrangements result in various three-dimensional nucleoprotein structures that differ in their shape and the capacity to constrain supercoils and compact the DNA. We believe that the DNA sequence-directed versatile assembly of periodic higher-order structures reveals a general organizational principle that can be exploited for knowledge-based design of long-range nucleoprotein complexes and purposeful manipulation of the bacterial chromatin architecture.

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