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. 2020 Dec 18;49(5):e30. doi: 10.1093/nar/gkaa1213

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Application of the screening assay to a DNA substrate. (A) The scheme of the Broccoli RNA fluorescence assay for high-throughput screening of AlkB activity towards O6mG in DNA. (B) The Broccoli DNA substrate with O6mG used in the assay. Conserved regions are labeled in green. O6mG is located at site 59. (C) The chemical structure of O6mG. (D) ssDNA with O6mG produced much lower level of fluorescence than the unmodified DNA. (E) Screening of the D135X library against O6mG identified positive hits. The absolute fluorescence signal generated with each member in the library was adjusted by subtracting it from the signal generated with the dead mutant. Variants with adjusted signals below zero were presented as zero to indicate that no activity was observed for these variants under the condition. (F) Reproducing positive signals of hits from the screening using the fluorescence assay. Error bar indicates SD, n ≥ 3. Statistical differences between the positive hits and the dead mutant were determined by Welch's t tests.