The existence of EML4–ALK, a mutated fusion gene in non-small-cell lung cancer (NSCLC), is an important consideration in the decision of the treatment options for NSCLC patients. However, there has been no standardized methods for EML4–ALK fusion gene detection up to now, which makes the development of novel detection probes particularly appealing. A molecular beacon is effective in detecting the level of a particular mRNA in living cells with easy signal visualization and convenient application. In this study, a molecular beacon composed of a gold nanoparticle core densely packed with FITC-labeled hairpin DNA sequences was synthesized and characterized. The presence of the EML4–ALK fusion gene will specifically open the hairpin structure, leading to the recovery of FITC fluorescence. This molecular beacon could precisely distinguish NSCLC cell lines with different levels of EML4–ALK mRNA expression, as indicated by obvious fluorescence in the EML4–ALK positive H2228 cell line and a negligible signal in the negative A549 cell line. The sensitivity and specificity of the molecular beacon were further verified in H2228 cells treated with small interfering RNAs (siRNAs) against ALK, which demonstrated decreased fluorescence after gene silencing. These results were simultaneously confirmed by flow cytometry and Q-PCR. More importantly, our molecular beacon successfully identified the EML4–ALK fusion gene in tissue specimens from 2 of 23 NSCLC patients. The results were consistent with Q-PCR quantification. Therefore, this molecular beacon holds great potential in clinical diagnosis of the EML4–ALK fusion gene with remarkable sensitivity and convenience.
Proteolysis-targeting chimeras (PROTACs) selectively eliminate detrimental proteins by exploiting the ubiquitin-proteasome system (UPS), representing a promising therapeutic strategy against various diseases. Effective adaptations of degradation signal sequences and E3 ligases for PROTACs remain limited. Here, we employed three amino acids─Gly, Pro, and Lys─as the ligand to recruit the corresponding E3 ligases: CRL2ZYG11B/ZER1, GID4, and UBRs, to degrade EML4-ALK and mutant EGFR, two oncogenic drivers in NSCLC. We found that the extent of EML4-ALK and EGFR reduction can be easily fine-tuned by using different degradation signals. These amino acid-based PROTACs, termed AATacs, hindered proliferation and induced cell cycle arrest and apoptosis of NSCLC cells in vitro. Compared to other PROTACs, AATacs are small, interchangeable but with different degradation efficiency. Our study further expands the repertoire of E3 ligases and their ligands for PROTAC application, improving the versatility and utility of targeted protein degradation for therapeutic purposes.
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