Lourenço Pedro, Cruz Carla
RISE-Health, Department of Medical Sciences, Faculty of Health Sciences, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
Department of Chemistry, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal.
Biomolecules. 2025 Mar 13;15(3):410. doi: 10.3390/biom15030410.
MicroRNA-155-3p (miR-155-3p) is an important biomarker in various pathological conditions, including cancer, making the development of sensitive and specific detection methods crucial. Here, we present a molecular beacon (MB-G4) that underwent a conformational switch upon hybridization with miR-155-3p, enabling the formation of a G-quadruplex (G4) structure. This G4 was recognized by the fluorogenic ligand N-methyl mesoporphyrin IX (NMM), producing a fluorescence signal proportional to the target concentration, making it a new detection method. The conformational dynamics of MB-G4 were characterized through circular dichroism (CD) spectroscopy and native polyacrylamide gel electrophoresis (PAGE), confirming the transition from a hairpin structure to an RNA-DNA hybrid duplex that facilitated G4 formation. The optimization of the experimental conditions, including the potassium chloride (KCl) and NMM concentrations, ensured selective detection with minimal background signal. The detection limit (LOD) was determined to be 10.85 nM, using a linear fluorescence response curve, and the specificity studies demonstrated a clear distinction between miR-155-3p and miR-155-5p. Furthermore, MB-G4 was studied with total RNA extracted from the lung cancer cell line A549 to evaluate its detection in a more complex environment and was able to detect its target, validating its potential for biological sample analysis.
微小RNA - 155 - 3p（miR - 155 - 3p）是包括癌症在内的各种病理状况下的重要生物标志物，因此开发灵敏且特异的检测方法至关重要。在此，我们展示了一种分子信标（MB - G4），它与miR - 155 - 3p杂交后会发生构象转换，从而能够形成一种G - 四链体（G4）结构。这种G4被荧光配体N - 甲基中卟啉IX（NMM）识别，产生与目标浓度成比例的荧光信号，使其成为一种新的检测方法。通过圆二色性（CD）光谱和非变性聚丙烯酰胺凝胶电泳（PAGE）对MB - G4的构象动力学进行了表征，证实了从发夹结构向促进G4形成的RNA - DNA杂交双链体的转变。包括氯化钾（KCl）和NMM浓度在内的实验条件的优化确保了以最小背景信号进行选择性检测。使用线性荧光响应曲线确定检测限（LOD）为10.85 nM，特异性研究表明miR - 155 - 3p和miR - 155 - 5p之间有明显区别。此外，用从肺癌细胞系A549提取的总RNA对MB - G4进行了研究以评估其在更复杂环境中的检测能力，并且能够检测到其靶标，验证了其在生物样品分析中的潜力。
