In this study, we successfully identified and validated an aptamer that can detect USE1, a significant biomarker for lung cancer. Validation was conducted using various methods, including Western blot, ELONA, and a Quencher DNA system, utilizing the USE1 protein, lung cancer cell lines, and lung cancer tissue samples. Additionally, we developed a STA-QD DNAMS fluorescence sensing method that effectively detects the USE1-targeting aptamer, leading to the creation of a diagnostic kit for lung cancer.
Lung cancer remains the most prevalent cancer and the second leading cause of cancer-related mortality worldwide [1, 2]. It can be cured via resection surgery if detected early[1, 2]. However, the lack of efficient early detection technologies or reliable molecular biomarkers often results in late diagnosis, thereby missing the ideal therapeutic window[3]. To improve patient survival, diagnostic technologies that use molecular biomarkers capable of sensitively and selectively recognizing lung cancer cells must be developed.
Previously, our research team discovered that the USE1 protein is upregulated in 98.1% of patients with lung cancer. The overexpression of the USE1 protein was found to promote the proliferation, migration, and invasion of lung cancer cells. Notably, USE1 contains a conserved D-box domain and is regulated by the anaphase-promoting complex, suggesting its pivotal role in the pathogenesis of lung cancer and highlighting its potential as a novel biomarker [8]. Based on these findings, we developed a practical diagnostic kit using aptamers that target the USE1 protein.
Aptamers are short single-stranded segments of DNA (ssDNA), RNA (ssRNA), or synthetic nucleic acid analogs (XNA) that bind their targets with high affinity and specificity by folding into various secondary and tertiary structures [11, 18]. Unlike antibodies, aptamers do not require immunogenicity to activate the host immune system, are synthesized chemically, and are easily modified and conjugated with other drugs or carriers [19]. Owing to these properties, aptamers have been extensively studied in various applications, including disease diagnosis, therapeutics, and biomarker discovery [10, 20].
Since the inception of Cell-SELEX for targeting lung cancer cells [21], several aptamers have been identified for the detection of lung cancer using lung cancer cell lines, tissue lysates, or patient blood samples. For instance, Zamay GS et al. [22] identified four aptamers that specifically bind to lung adenocarcinoma cells using blood circulating tumor cells (CTCs). These aptamers were applied not only to detect CTCs and apoptotic bodies but also to associated protein biomarkers for lung cancer, including vimentin, annexin A2, annexin A5, histone 2B, neutrophil defensin, and clusterin. Zhou et al. [23] identified a novel biomarker for small-cell lung cancer (SCLC) through Cell-SELEX-generated aptamers; aptamer C12 was found to target high-density lipoprotein binding protein (HDLBP). Subsequent knockdown of HDLBP by siRNA inhibited the proliferation and metastasis of SCLC cells in vitro and decelerated tumor formation in vivo. More recently, the AP-9R aptamer was developed using Cell-SELEX to target cancer stem cells of lung cancer, with annexin A2 identified as the target protein. The expression of annexin A2 was associated with stemness, metastasis, and poor clinical outcomes in lung cancer, suggesting its role as a cancer stem cell (CSC) marker and regulator, with potential theragnostic applications for lung cancer [24]. These aptamers, which target lung cancer cell lines, tissue lysates, and patient serum, were validated as detectors of biomarkers by identifying the target proteins and assessing the impact on lung cancer development or progression. However, the specificity and sensitivity of these aptamers may be compromised as they do not commence from the exact target molecule and might inadvertently detect other cancers or normal molecules.
In contrast to previous studies, the present study aimed to develop a lung cancer detection kit using an aptamer that specifically targets the USE1 protein, which is already well-studied due to its role as a novel biomarker and its involvement in lung cancer pathogenesis. By directly targeting USE1, our approach aims to enhance the sensitivity and specificity of lung cancer detection. This strategic focus is expected to yield a diagnostic tool that not only improves the accuracy of lung cancer diagnostics but also contributes to earlier and more effective treatment interventions.
Among the three aptamers developed in this study, aptamer 1 exhibited the best binding affinity to USE1, with a dissociation constant of 10 nM. To reduce synthesis costs and improve tissue penetration and structural stability, aptamer 1 was truncated to aptamer 1e, which demonstrated similarly high binding affinity. Aptamers 1 and 1e effectively interacted with USE1 in vitro and in vivo based on immunoprecipitation assays and could identify lung cancer tissues with 100% sensitivity and 80% specificity based on ELONA. These results confirm that aptamers 1 and 1e are effective detectors of the USE1 protein not only in vitro and in lung carcinoma cell lines but also in actual patient cancer tissues, underscoring their potential for clinical application in lung cancer diagnostics.
RCA is a method in which nucleic acids are synthesized using a circular template [25]. This technique is not only used to amplify and subsequently detect analyte molecules but also to generate functionally active nucleic acids that mediate the detection of other biotargets [26]. Self-assembled DNAMS was produced via RCA with several functionalized dNTPs, which can exhibit DNAMS fluorescence and allow the attachment of other functional moieties through strong interactions, such as chemical conjugation or biotin–streptavidin interaction [25, 26].
Building on this technology, we developed a USE1-targeting aptamer detection kit using self-assembled biotin-modified DNAMS and STA-QD conjugation. This kit enables successful identification of the USE1-associated aptamer 1 through simple visual inspection, offering a promising tool for early and effective lung cancer diagnosis. This integration of STA-QD DNAMS with a USE1-targeted aptamer implies the potential for sophisticated diagnostic applications in lung cancer, potentially improving clinical outcomes through early detection.
In this study, aptamers targeting USE1, a critical biomarker for lung cancer, were successfully developed and validated. Aptamer 1 and its truncated form, Aptamer 1e, exhibited high binding affinity and specificity to USE1, demonstrating effectiveness in various in vitro assays and in distinguishing lung cancer tissues from non-tumoral samples. The integration of these aptamers into a newly formulated diagnostic kit, utilizing RCA-generated, biotin-modified DNA microstructures with STA-QD conjugation, marks a significant advancement in lung cancer diagnostics. This kit, designed for simple visual inspection, is characterized by high sensitivity and specificity, and has the potential to substantially enhance the early detection and treatment of lung cancer. Overall, this approach could be transformative, setting a new standard for biomarker-based diagnostics and potentially decreasing the mortality rate of lung cancer through earlier intervention.