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目的:观察分子信标(molecular beacons,MB)在活细胞内及在体水平对内源性m RNA检测的可行性。方法:合成增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,e GFP)的MB,模拟体内条件,分别从MB的稳定性、与目标基因结合的特异性和敏感性进行检测,并通过在活细胞和斑马鱼胚胎内的应用来观察其对内外源性m RNA的检测的可行性。结果:MB在37℃条件下在体外48小时内经琼脂糖凝胶电泳鉴定未见降解,熔解曲线结果显示荧光强度差异无统计学意义(P>0.05);并且只有在靶序列存在的条件下MB的信号强度显著增强,MB的信号强度与靶基因浓度呈正相关并能基本稳定存在;经e GFP和靶向e GFP的MB共转染的293T细胞能看到荧光表达,靶向e GFP的MB可特异性结合细胞内e GFP的m RNA;在单细胞期斑马鱼胚胎内共注射MB以及目标单链后能观察到荧光表达。结论:结果表明分子信标可用于活细胞内内源性m RNA的检测。
Objective: To observe the feasibility of detecting molecular endogenous m RNA in molecular beacons (MB) in living cells and in vivo. Methods: MBs of Enhanced Green Fluorescent Protein (eGFP) were synthesized and their in vivo conditions were mimicked. The stability and the specificity and sensitivity of the target genes were examined respectively. The application of zebrafish embryos to observe the detection of endogenous and exogenous m RNA is feasible. Results: The degradation of MB was not observed by agarose gel electrophoresis in 48 hours in vitro at 37 ℃. The results of melting curve showed no significant difference in fluorescence intensity (P> 0.05). MB only existed in the presence of target sequence The signal intensity of MB was positively correlated with the concentration of target gene and could be basically stable. The expression of eGFP-targeted 293T cells transfected with eGFP and MB targeting eGFP Can specifically bind intracellular e GFP m RNA; fluorescent expression was observed after co-injection of MB and target single strand in a single-cell zebrafish embryo. Conclusion: The results show that molecular beacons can be used to detect endogenous m RNA in living cells.