Quantification of dye-mediated photodamage during single-molecule DNA imaging

doi:10.1016/j.ab.2012.03.021

Analytical Biochemistry

Volume 426, Issue 1, 1 July 2012, Pages 13-21

https://doi.org/10.1016/j.ab.2012.03.021 Get rights and content

Abstract

Single-molecule fluorescence imaging of DNA-binding proteins has enabled detailed investigations of their interactions. However, the intercalating dyes used to visually locate DNA molecules have the undesirable effect of photochemically damaging the DNA through radical intermediaries. Unfortunately, this damage occurs as single-strand breaks (SSBs), which are visually undetectable but can heavily influence protein behavior. We investigated the formation of SSBs on DNA molecules by the dye YOYO-1 using complementary single-molecule imaging and gel electrophoresis-based damage assays. The single-molecule assay imaged hydrodynamically elongated lambda DNA, enabling the real-time detection of double-strand breaks (DSBs). The gel assay, which used supercoiled plasmid DNA, was sensitive to both SSBs and DSBs. This enabled the quantification of SSBs that precede DSB formation. Using the parameters determined from the gel damage assay, we applied a model of stochastic DNA damage to the time-resolved DNA breakage data, extracting the rates of single-strand breakage at two dye staining ratios and measuring the damage reduction from the radical scavengers ascorbic acid and β-mercaptoethanol. These results enable the estimation of the number of SSBs that occur during imaging and are scalable over a wide range of laser intensities used in fluorescence microscopy.

Section snippets

Materials and methods

All chemicals and materials were Fisher brand unless otherwise noted.

Double-strand photocleavage of individual DNA molecules

We applied SMI to observe double-strand photocleavage of individual dye-labeled DNA molecules over time. Lambda DNA molecules were end-labeled with biotin, stained with YOYO-1 intercalating dye, and injected into a microfluidic flow cell whose surfaces had been functionalized with a PEG/biotin–PEG layer. The PEG coating minimizes nonspecific electrostatic interactions between the DNA and the glass, whereas the relatively small subpopulation of biotin-terminated PEG provides binding sites for

Discussion

The purpose of our study was to quantify single-strand photocleavage during SMI experiments because this form of damage is not readily apparent. We do note that the SMI DNA breakage curves all share the common feature of an initial plateau before decaying (Fig. 1B). This plateau corresponds to the induction period during which SSBs are accumulating but are insufficient in frequency to cause double-strand cleavage. The stochastic DNA damage model used to fit the data captures this feature (which

Conclusion

This work was undertaken to quantify the damage mediated by common fluorescent DNA intercalaters on DNA substrates during imaging experiments. Although we tested YOYO-1, these findings are applicable to any DNA intercalater, including those used to image nucleic acids in live cells. We determined the breakage rates of DNA using a gel-based assay, gaining information about the separation of strand breaks required to linearize the molecule and the ability of radical scavengers to reduce damage

Acknowledgments

The authors thank the University of North Carolina and the National Science Foundation for the Duke Energy BioSciences Scholar (DEBS) and Science and Math Achievement and Resourcefulness Track (SMART) programs, which made their research possible.

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Quantification of dye-mediated photodamage during single-molecule DNA imaging

Abstract

Section snippets

Materials and methods

Double-strand photocleavage of individual DNA molecules

Discussion

Conclusion

Acknowledgments

Anal. Biochem.

Mol. Cell

J. Biochem. Biophys. Methods

J. Theor. Biol.

J. Biol. Chem.

See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins

Nucleic Acids Res.

Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase

Nature

Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

Nature

Physical mechanisms of generation and deactivation of singlet oxygen

Chem. Rev.

Radiation-induced DNA damage and its repair

Int. J. Radiat. Biol.

The yield of DNA double strand breaks produced intracellularly by ionizing radiation: a review

Int. J. Radiat. Biol.

Gapped DNA is anisotropically bent

Proc. Natl. Acad. Sci. USA

Single- and double-strand break formation in DNA irradiated in aqueous solution: dependence on dose and OH radical scavenger concentration

Radiat. Res.