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High-Fidelity and Simultaneous Sensing of Endogenous Mutant and Wild p53 Proteins for Precise Cancer Diagnosis and Drug Screening.

Fang YangXia LiRuo YuanYun Xiang
Published in: Analytical chemistry (2021)
The simultaneous sensing of endogenous wild and mutant proteins plays a critical role in disease diagnosis and drug screening, and this remains a major current challenge. Here, we present a new and highly specific target-triggered dual proximity ligation assay (dPLA) strategy for sensitive and simultaneous sensing of wild and mutant p53 proteins from cancer cells. Two proximity DNA probes bind the target protein to form the primer/circular DNA template complexes with two nicks in the presence of the hairpin and ssDNA connector sequences via the strand displacement reaction. Only when the two nicks are simultaneously ligated can the rolling circle amplification be triggered with high fidelity for yielding substantially enhanced fluorescence. By encoding the hairpin sequence, two distinct fluorescence signals can be generated for simultaneous detection of the wild and mutant p53 proteins. Importantly, our method significantly reduces the possibility of nonspecific ligation reactions by using two ligation nicks, which minimizes the background noise. With this dPLA method, the regulation transition of intracellular mutant p53 to wild p53 proteins upon anticancer drug treatment has also been demonstrated, highlighting its usefulness for potential early disease diagnosis and drug screening with high fidelity.
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