Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131, Karlsruhe, Germany.
Karlsruhe Institute of Technology (KIT), Institute of Applied Physics, Wolfgang-Gaede-Str. 1, 76131, Karlsruhe, Germany.
Chembiochem. 2021 Aug 3;22(15):2561-2567. doi: 10.1002/cbic.202100150. Epub 2021 Jun 25.
For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2'-positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by fluorescence lifetime imaging microscopy (FLIM), which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of GFP expression, which demonstrates the potential of our siRNA architectures as a tool for future development of effective siRNA.
为了监测小干扰 RNA（siRNA）的细胞内途径，我们通过两个 ATTO 染料在内部位置标记了两条链，作为链间Förster 共振能量转移对。siRNA 双链显示红色发射和短供体寿命作为读出，而 siRNA 反义单链显示绿色发射和长供体寿命。如果可以预期 GFP 沉默（绿色），则发出此读出信号，否则发出红色信号。我们通过后期合成修饰将两种染料附着到三个结构不同的炔烃锚上。染料在 2'-位置时，核糖呋喃糖苷锚仅有轻微偏好。首次通过荧光寿命成像显微镜（FLIM）跟踪了 siRNA 在活 HeLa 细胞中的递呈和命运，这揭示了使用不同转染方法的细胞内运输与 GFP 表达敲低之间的明确关系，这表明我们的 siRNA 结构具有作为未来有效 siRNA 开发工具的潜力。
