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. 2012 Feb 13;51(7):1689-92.
doi: 10.1002/anie.201108135. Epub 2012 Jan 12.

Direct fluorescence monitoring of DNA base excision repair

Toshikazu Ono  1 , Shenliang Wang, Chi-Kin Koo, Lisa Engstrom, Sheila S David, Eric T Kool
Affiliations

Direct fluorescence monitoring of DNA base excision repair

Toshikazu Ono et al. Angew Chem Int Ed Engl. .
No abstract available

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Figures

Figure 1
Figure 1
A) Structure of probe 1; B) Sequences of probes in this study. Y = α-pyrene deoxyriboside; S = tetrahydrofuran spacer
Figure 2
Figure 2
In vitro enzymatic assay of probes incubated with E. coli UDG. (A) Fluorescence spectra over a 60-min timecourse (excitation 340 nm). (B) Increase in emission over time at 395 nm, along with data for thymine-containing control (2) and with UDG inhibitor (UGI). [probe] = 400 nM; [UDG] = 1 unit/mL; [UGI] = 1 unit/mL at pH 8.0, 37 °C.
Figure 3
Figure 3
MALDI-TOF mass spectra of (A) probe 1, (B) probe 1 after treatment with UDG, (C) probe 3 and (D) probe 3 after treatment with UDG, confirming loss of uracil in the enzymatic reaction.
Figure 4
Figure 4
Images and spectra of sensors with bacterial cells. (A,B) Bright field (left) and fluorescence microscope images (right) of Escherichia coli (BW(310)DE3udg(-)) cells transformed with the pET28a plasmid containing the Afu UDG gene. Cells were incubated in a solution containing (A) probe 1 or (B) thymine control probe 2 at 5 μM for 4 h (65 °C). Excitation 330-380 nm; emission >420 nm. (C,D) Spectra of bacterial solutions diluted tenfold (excitation 340 nm). (C) Probe 1 with bacteria expressing Afu UDG, incubated with probe for 1 or 4 h; (D) Control probe 2 with bacteria under the same conditions.
See this image and copyright information in PMC

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Publication types