Chen Antony K, Behlke Mark A, Tsourkas Andrew
Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Nucleic Acids Res. 2008 Jul;36(12):e69. doi: 10.1093/nar/gkn331. Epub 2008 May 24.
Fluorescent microscopy experiments show that when 2'-O-methyl-modified molecular beacons (MBs) are introduced into NIH/3T3 cells, they elicit a nonspecific signal in the nucleus. This false-positive signal can be avoided by conjugating MBs to macromolecules (e.g. NeutrAvidin) that prevent nuclear sequestration, but the presence of a macromolecule makes efficient cytosolic delivery of these probes challenging. In this study, we explored various methods including TAT peptide, Streptolysin O and microporation for delivering NeutrAvidin-conjugates into the cytosol of living cells. Surprisingly, all of these strategies led to entrapment of the conjugates within lysosomes within 24 h. When the conjugates were pegylated, to help prevent intracellular recognition, only microporation led to a uniform cytosolic distribution. Microporation also yielded a transfection efficiency of 93% and an average viability of 86%. When cells microporated with MB-NeutrAvidin conjugates were examined via flow cytometry, the signal-to-background was found to be more than 3 times higher and the sensitivity nearly five times higher than unconjugated MBs. Overall, the present study introduces an improved methodology for the high-throughput detection of RNA at the single cell level.
荧光显微镜实验表明，当将2'-O-甲基修饰的分子信标（MBs）引入NIH/3T3细胞时，它们会在细胞核中引发非特异性信号。通过将MBs与防止核隔离的大分子（如中性抗生物素蛋白）偶联，可以避免这种假阳性信号，但大分子的存在使得这些探针有效地递送至细胞质具有挑战性。在本研究中，我们探索了多种方法，包括TAT肽、链球菌溶血素O和微孔导入法，用于将中性抗生物素蛋白偶联物递送至活细胞的细胞质中。令人惊讶的是，所有这些策略在24小时内都会导致偶联物被困在溶酶体内。当偶联物进行聚乙二醇化以帮助防止细胞内识别时，只有微孔导入法能导致其在细胞质中均匀分布。微孔导入法还产生了93%的转染效率和86%的平均存活率。当通过流式细胞术检测用MB-中性抗生物素蛋白偶联物进行微孔导入的细胞时，发现信号与背景的比值比未偶联的MBs高出3倍以上，灵敏度高出近5倍。总体而言，本研究介绍了一种用于单细胞水平RNA高通量检测的改进方法。
