Nitin Nitin, Santangelo Philip J, Kim Gloria, Nie Shuming, Bao Gang
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Nucleic Acids Res. 2004 Apr 14;32(6):e58. doi: 10.1093/nar/gnh063.
Real-time visualization of specific endogenous mRNA expression in vivo has the potential to revolutionize medical diagnosis, drug discovery, developmental and molecular biology. However, conventional liposome- or dendrimer-based cellular delivery of molecular probes is inefficient, slow, and often detrimental to the probes. Here we demonstrate the rapid and sensitive detection of RNA in living cells using peptide-linked molecular beacons that possess self-delivery, targeting and reporting functions. We conjugated the TAT peptide to molecular beacons using three different linkages and demonstrated that, at relatively low concentrations, these molecular beacon constructs were internalized into living cells within 30 min with nearly 100% efficiency. Further, peptide-based delivery did not interfere with either specific targeting by or hybridization-induced fluorescence of the probes. We could therefore detect human GAPDH and survivin mRNAs in living cells fluorescently, revealing intriguing intracellular localization patterns of mRNA. We clearly demonstrated that cellular delivery of molecular beacons using the peptide-based approach has far better performance compared with conventional transfection methods. The peptide-linked molecular beacons approach promises to open new and exciting opportunities in sensitive gene detection and quantification in vivo.
体内特定内源性mRNA表达的实时可视化有潜力彻底改变医学诊断、药物研发、发育生物学和分子生物学。然而，基于传统脂质体或树枝状大分子的分子探针细胞递送效率低下、速度缓慢，且常常对探针有害。在此，我们展示了使用具有自我递送、靶向和报告功能的肽连接分子信标对活细胞中的RNA进行快速灵敏检测。我们使用三种不同的连接方式将TAT肽与分子信标偶联，并证明在相对较低的浓度下，这些分子信标构建体在30分钟内以近100%的效率内化到活细胞中。此外，基于肽的递送既不干扰探针的特异性靶向，也不干扰杂交诱导的荧光。因此，我们能够在活细胞中荧光检测人GAPDH和存活素mRNA，揭示了有趣的mRNA细胞内定位模式。我们清楚地证明，与传统转染方法相比，使用基于肽的方法进行分子信标的细胞递送具有更好的性能。肽连接分子信标方法有望在体内灵敏基因检测和定量方面开创令人兴奋的新机遇。
