Endonucleolytic inhibition assay of DNA/Fok I transducer as a sensitive platform for homogeneous fluorescence detection of small molecule-protein interactions.
This paper reported a novel homogeneous fluorescence assay strategy for probing small molecule-protein interactions based on endonucleolytic inhibition of a DNA/Fok I transducer. The transducer could cyclically cleave fluorescence-quenched probes to yield activated fluorescence signal, while protein binding to the small molecule label would prevent Fok I from approaching and cleaving the fluorescence-quenched probes. Because of the efficient signal amplification from the cyclic cleavage operation, the developed strategy could offer high sensitivity for detecting small molecule-protein interactions. This strategy was demonstrated using folate and its high-affinity or low-affinity binding proteins. The results revealed that the developed strategy was highly sensitive for detecting either high- or low-affinity small molecule-protein interactions with improved selectivity against nonspecific protein adsorption. This strategy could also be extended for assays of candidate small-molecule ligands using a competitive assay format. Moreover, this strategy only required labeling the small molecule on a DNA heteroduplex, circumventing protein modifications that might be harmful for activity. In view of these advantages, this new method could have potential to become a universal, sensitive, and selective platform for quantitative assays of small molecule-protein interactions.
State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha, P R China.
Zhen
Z
Zhen
Li-Juan
LJ
Tang
Jian
J
Lin
Jian-Hui
JH
Jiang
Ru-Qin
RQ
Yu
Xiangling
X
Xiong
Weihong
W
Tan
D000818:Animals; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014159:Transducers
D005456:Fluorescent Dyes; D008024:Ligands; D011506:Proteins; D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific