van der Wiel Ingrid M, Cheng Jenny, Koukiekolo Roger, Lyn Rodney K, Stevens Nathan, O'Connor Naphtali, Turro Nicholas J, Pezacki John Paul
Steacie Institute for Molecular Sciences, National Research Council, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6.
J Am Chem Soc. 2009 Jul 29;131(29):9872-3. doi: 10.1021/ja902636m.
Here we report that the phenanthridine derivative covalently linked to a fluorescein moiety (FLEth) can act as a fluorescence based probe for duplex short interfering RNA (siRNA) and that this probe can also be used to report on protein-RNA interactions. A fluorescence resonance energy transfer (FRET) signal that is observed at 600 nm occurs when FLEth is complexed with siRNA. At least 2 molecules of FLEth can bind to 21 nt duplex siRNA, and the dissociation constants for these interactions are reported. We find that FLEth can also report on the interaction of siRNAs with the Carnation Italian ringspot viral suppressor of RNA silencing p19. FLEth does not bind to the siRNA-p19 complex nor can p19 bind to the siRNA-FLEth complex; rather FLEth can report on the fraction of siRNA that is unbound. FLEth can also bind siRNA in delivery systems such as liposomes. Once the siRNA reaches the interior of Huh 7.5 cells, FLEth dissociates from the siRNA and is found in the nucleoli suggesting that FLEth cannot bind to siRNAs that are associated with the RNA silencing machinery.
在此我们报告，与荧光素部分共价连接的菲啶衍生物（FLEth）可作为基于荧光的双链短干扰RNA（siRNA）探针，并且该探针还可用于报告蛋白质-RNA相互作用。当FLEth与siRNA复合时，会在600nm处观察到荧光共振能量转移（FRET）信号。至少2个FLEth分子可与21个核苷酸的双链siRNA结合，并报告了这些相互作用的解离常数。我们发现FLEth还可报告siRNA与康乃馨意大利环斑病毒RNA沉默抑制因子p19的相互作用。FLEth不与siRNA-p19复合物结合，p19也不与siRNA-FLEth复合物结合；相反，FLEth可报告未结合的siRNA的比例。FLEth还可在脂质体等递送系统中与siRNA结合。一旦siRNA到达Huh 7.5细胞内部，FLEth就会从siRNA上解离，并在核仁中被发现，这表明FLEth不能与与RNA沉默机制相关的siRNA结合。
