Journal
BIOSENSORS & BIOELECTRONICS
Volume 98, Issue -, Pages 210-214Publisher
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2017.06.052
Keywords
Pyrrolo-dC; 8-oxoguanine; Base excision repair; Formamidopyrimidine DNA glycosylase; Enzyme activity assay; Fluorescent biosensor
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Funding
- Mid-career Researcher Support Program through the National Research Foundation (NRF) - Ministry of Science, ICT & Future Planning (MSIP) of Korea [2015R1A2A1A01005393]
- Center for BioNano Health Guard - MSIP of Korea as Global Frontier Project [H-GUARD_2013M3A6B2078964]
- NRF grant - MSIP of Korea [2017R1C1B5017724]
- National Research Foundation of Korea [2017R1C1B5017724] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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We develop a novel approach to determine formamidopyrimidine DNA glycosylase (Fpg) activity by taking advantage of the unique fluorescence property of pyrrolo-dC (PdC) positioned opposite to 8-oxoguanine (8-oxoG) in duplex DNA. In its initial state, PdC in duplex DNA undergoes the efficient stacking and collisional quenching interactions, showing the low fluorescence signal. In contrast, the presence of Fpg, which specifically removes 8-oxoG and incises resulting apurinic (AP) site, transforms duplex DNA into single-stranded (ss) DNAs. As a result, the intrinsic fluorescence signal of PdC in ssDNA is recovered to exhibit the significantly enhanced fluorescence signal. Based on this Fpg-dependent fluorescence response of PdC, we could reliably determine Fpg activity down to 1.25 U/ml with a linear response from 0 to 50 U/ml. In addition, the diagnostic capability of this strategy was successfully demonstrated by reliably assaying Fpg activity in human blood serum, showing its great potential in the practical applications.
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