Lifland Aaron W, Zurla Chiara, Santangelo Philip J
Georgia Institute of Technology and Emory University, Atlanta, Georgia.
Bioconjug Chem. 2010 Mar 17;21(3):483-8. doi: 10.1021/bc9003876. Epub 2010 Feb 8.
The imaging of RNA in live cells presents a methodological challenge both in the mechanism by which the probes are delivered to the cell and in the sensitivity of the probe. Probes must be delivered to the correct cellular compartment and, once inside the cell, should bind to the target RNA rapidly and with enough sensitivity to detect small numbers of RNA molecules. Here, we report the characterization of a single-molecule sensitive, multivalent RNA imaging probe that utilizes an eight-armed poly(ethylene glycol) core. This probe allowed for the accurate imaging of the human respiratory syncytial virus (hRSV) genomic RNA when delivered into live cells using either reversible membrane permeabilization or TAT-peptide mediated membrane transduction. Neither reversible membrane permeabilization nor TAT mediated delivery proceeded through the endosomal pathway, and both delivery methods demonstrated fast binding kinetics of less than 10 min. Subcellular distributions of RNA were visualized in live cells, as well as RNA-protein colocalization, after fixation and immunostaining of the hRSV RNA binding protein N.
活细胞中RNA的成像在探针递送至细胞的机制以及探针的灵敏度方面都面临着方法学上的挑战。探针必须递送至正确的细胞区室，并且一旦进入细胞，就应快速且以足够的灵敏度与靶RNA结合，以检测少量的RNA分子。在此，我们报道了一种利用八臂聚乙二醇核心的单分子敏感多价RNA成像探针的特性。当使用可逆膜通透化或TAT肽介导的膜转导将该探针递送至活细胞时，它能够对人呼吸道合胞病毒（hRSV）基因组RNA进行精确成像。可逆膜通透化和TAT介导的递送均不通过内体途径，并且两种递送方法均显示出小于10分钟的快速结合动力学。在对hRSV RNA结合蛋白N进行固定和免疫染色后，在活细胞中观察到了RNA的亚细胞分布以及RNA-蛋白质共定位。
