Department of Chemical Biology
Yang's Lab,Department of Chemical Biology, Xiamen University
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Professor Yang

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Yang's Laboratory

Room 532, Lujiaxi Building, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China

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Mingxuan Gao's paper has been accepted by ACS APPLIED Materials and Interfaces

2017-10-16 03:18:51

As a vital enzyme in DNA phosphorylation and restoration, T4 polynuceotide kinase (T4 PNK) has aroused great interest in recent years. Therefore, numerous strategies have been established for highly sensitive detection of T4 PNK based on diverse signal amplification techniques. However, they often need sophisticated design, a variety of auxiliary reagents and enzymes, or cumbersome manipulations. We have designed a new kind of allosteric aptamer probe consisting of streptavidin aptamer (SA aptamer) and the complementary DNA (cDNA) for simple detection of T4 PNK without signal amplification and with minimized interference in complex biological samples. When the 5’-terminus of the cDNA is phosphorylated by T4 PNK, the cDNA is degraded by Lambda exonuclease (λ exo) to release the FAM-labelled SA aptamer, which subsequently binds to streptavidin beads (SA beads). The enhancement of the fluorescence signal on SA beads can be detected precisely and easily by a microscope or flow cytometer. Our method performs well in complex biological samples as a result of the enrichment of the signaling molecules on beads, as well as simple manipulations to discard the background interference and non-binding molecules. Without signal amplification techniques, our allosteric aptamer probe method not only avoids complicated manipulations but also decreases the time required. With the advantages of ease of operation, reliability and robustness for T4 PNK detection in buffer as well as real biological samples, the allosteric aptamer probe has great potential for clinical diagnostics, inhibitor screening and drug discovery.