FLEth RNA intercalating probe is a convenient reporter for small interfering RNAs.
FLEth RNA intercalating probe is a convenient reporter for small interfering RNAs.
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DOI：
10.1021/ja902636m
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发表时间：
2009-07-29
影响因子：
15
通讯作者：
Pezacki, John Paul
中科院分区：
文献类型：
--
作者：
van der Wiel, Ingrid M.;Cheng, Jenny;Koukiekolo, Roger;Lyn, Rodney K.;Stevens, Nathan;O'Connor, Naphtali;Turro, Nicholas J.;Pezacki, John Paul
在这里，我们报道了与荧光素部分共价连接的菲啶衍生物(FLETH)可以作为基于荧光的双链短干扰RNA的探针，该探针也可以用于报告蛋白质−RNA的相互作用。当FLEth与siRNA络合时，在600 nm处观察到荧光共振能量转移(FRET)信号。至少有2个FLETH分子可以与21个核苷酸双链siRNA结合，并报道了这些相互作用的解离常数。我们发现FLEth还可以报告siRNAs与香石竹意大利环斑病毒抑制RNA沉默p19的相互作用。FLEth不与siRNA−p19复合体结合，也不能与siRNA−FLEth复合体结合；相反，FLEth可以报告未结合的siRNA的部分。FLEth还可以结合脂质体等递送系统中的siRNA。一旦siRNA到达Huh 7.5细胞的内部，FLEth就从siRNA中解离出来，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。
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.
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