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Photochemical determination of different DNA structures

Nature Protocols volume 2, pages 78–87 (2007)Cite this article

Abstract

The various conformations of DNA are thought to have important biological roles. Investigation of the local DNA conformational changes associated with biological events is therefore essential to an understanding of the functions of DNA. We have reported the photoreactivities of 5-halouracil in the five characteristic local DNA structures: the A, B and Z forms, protein-induced DNA kinks and the G-quadruplex form. These studies demonstrate the detailed relationships between the local DNA structures and the photochemical products of photoinduced hydrogen abstraction by the resulting uracil-5-yl radicals, and show that this photochemical method can be used to detect DNA structures. Here, we describe in detail procedures that have been developed in our laboratory for probing DNA conformations by product analysis of photoirradiated 5-halouracil–containing DNA. The protocol includes the preparation of 5-halouracil–containing DNA and the characterization of the photoproducts, and it can be completed in 2 weeks.

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Figure 1
Figure 2: Flow scheme of the various procedures involved in the product analysis for the five different structures.
Figure 3
Figure 4: The tryptophan residue of Sso7d is efficiently oxidized to N′-formylkynulenine (NFK).
Figure 5
Figure 6
Figure 7: C1′- and C2′α-hydrogen abstraction in B-DNA.
Figure 8: Stereospecific C2′α-hydroxylation in Z-DNA (a) HPLC profiles of UV-irradiated ODN4/ODN5 in the presence of Zα.
Figure 9: Photochemical reaction in protein-induced DNA kink.
Figure 10: Highly efficient photochemical formation of C2′-deoxyribonolacton in the antiparallel G-quadruplex.

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Acknowledgements

This study was partly supported by a Grant-in-Aid for Priority Research from the Ministry of Education, Science, Sports, and Culture and a grant from Solution Oriented Research for Science and Technology of Japan Science and Technology.

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  1. Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, 606-8502, Kyoto, Japan

    Yan Xu, Ryu Tashiro & Hiroshi Sugiyama

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Correspondence to Hiroshi Sugiyama.

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Xu, Y., Tashiro, R. & Sugiyama, H. Photochemical determination of different DNA structures. Nat Protoc 2, 78–87 (2007). https://doi.org/10.1038/nprot.2006.467

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