A sensitive label-free fluorescence assay for monitoring T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on a coupled λ exonuclease cleavage reaction and SYBR Green I. In this assay, a double-stranded DNA (dsDNA) was stained with SYBR Green I and used as a substrate for T4 PNK. After the 5′-hydroxyl termini of the dsDNA was phosphorylated by the T4 PNK, the coupled λ exonuclease began to digest the dsDNA to form mononucletides and single-stranded DNA (ssDNA). At this moment, the fluorescence intensity of the SYBR Green I decreased because of less affinity with ssDNA than dsDNA. The decreasing extent was proportional to the concentration of the T4 PNK. After optimization of the detection conditions, including the concentration of ATP, amount of λ exonuclease and reaction time, the activity of T4 PNK was monitored by the fluorescence measurement, with the limit of detection of 0.11 U mL −1 and good linear correlation between 0.25–1.00 U mL −1 ( R 2 = 0.9896). In this assay, no label was needed for fluorescence detection. Moreover, the inhibition behaviors of the T4 PNK's inhibitors were evaluated by this assay. The result indicated the potential of using this assay for monitoring of the phosphorylation-related process.
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A graphene oxide-based fluorescence assay for the sensitive detection of DNA exonuclease enzymatic activity
The 3′–5′ exonuclease enzyme plays a dominant role in multiple pivotal physiological activities, such as DNA replication and repair processes. In this study, we designed a sensitive graphene oxide (GO)-based probe for the detection of exonuclease enzymatic activity. In the absence of Exo III, the strong π–π interaction between the fluorophore-tagged DNA and GO causes the efficient fluorescence quenching via a fluorescence resonance energy transfer (FRET). In contrast, in the presence of Exo III, the fluorophore-tagged 3′-hydroxyl termini of the DNA probe was digested by Exo III to set the fluorophore free from adsorption when GO was introduced, causing an inefficient fluorescence quenching. As a result, the fluorescence intensity of the sensor was found to be proportional to the concentration of Exo III; towards the detection of Exo III, this simple GO-based probe demonstrated a highly sensitive and selective linear response in the low detection range from 0.01 U mL −1 to 0.5 U mL −1 and with the limit of detection (LOD) of 0.001 U mL −1 . Compared with other fluorescent probes, this assay exhibited superior sensitivity and selectivity in both buffer and fetal bovine serum samples, in addition to being cost effective and having a simple setup.
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- Award ID(s):
- 1709160
- NSF-PAR ID:
- 10161040
- Date Published:
- Journal Name:
- The Analyst
- Volume:
- 144
- Issue:
- 21
- ISSN:
- 0003-2654
- Page Range / eLocation ID:
- 6231 to 6239
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/c9an01283d
